CN114149510B - Condition-controlled splicing chimeric antigen receptor molecule and application thereof - Google Patents

Condition-controlled splicing chimeric antigen receptor molecule and application thereof Download PDF

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CN114149510B
CN114149510B CN202111273025.5A CN202111273025A CN114149510B CN 114149510 B CN114149510 B CN 114149510B CN 202111273025 A CN202111273025 A CN 202111273025A CN 114149510 B CN114149510 B CN 114149510B
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CN114149510A (en
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徐建青
张晓燕
廖启彬
丁相卿
王诗语
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Shanghai Sinobay Bio Tech Co ltd
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Abstract

The invention provides a conditional control spliceable chimeric antigen receptor molecule and application thereof, wherein the spliceable chimeric antigen receptor molecule comprises an antigen recognition unit and a signal transduction unit, and the antigen recognition unit comprises an antigen recognition domain, a transmembrane domain, a co-stimulatory signal domain, an N-terminal splicing domain and a degradation agent; the signal transduction unit comprises a conditional signal response domain, a C-terminal splice domain, and a signaling domain. Such a conditionally controlled alternative splicing system allows splicing of two units and signaling under tumor microenvironment signals. Wherein the antigen recognition unit can spontaneously/evoke degradation, reducing retention in normal tissue. The signal transmission unit can respond to the signals of the specific conditions of the tumor microenvironment, and has the characteristics of low expression in the normal tissue environment and high expression in the tumor microenvironment. The conditional control spliceable system can realize the preparation and accurate treatment of solid tumor therapeutic drugs by accessing different efficacy genes.

Description

Condition-controlled splicing chimeric antigen receptor molecule and application thereof
Technical Field
The invention belongs to the technical field of biological medicine, and in particular relates to a conditional control splicing chimeric antigen receptor molecule, a coding nucleic acid molecule, a vector and a host cell thereof, and application thereof in treating anaerobic diseases.
Background
Cancer immunotherapy involves therapies such as monoclonal antibodies, vaccines, gene therapy, cell therapy, etc., wherein monoclonal antibodies targeting tumor antigens, immune checkpoint inhibitors, T cell receptor modified T cells (T cell receptor T cells, TCR-T cells), chimeric antigen receptor T cells (Chimeric antigen receptor T cells, CAR-T cells), etc., have shown remarkable antitumor effects in clinical studies. However, in the context of cell therapy, off-target effects due to the lack of tumor-specific antigens often can cause fatal side effects (Morgan RA, yang JC, kitano M, et al Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB [ J ]. Mol Ther.2010, 18:843-851), thereby greatly limiting the clinical use of cell therapy in solid tumors. Therefore, there is a need to further develop a controllable cellular immunotherapy technique with space-time specificity, which can reduce the damage to normal tissues and improve the treatment specificity to tumor lesions.
Previous studies reported a modular CAR design, where researchers split the complete CAR molecule into two modules, respectively antigen recognition modules located on the cell membrane, which are used to recognize antigens on the tumor cell surface; the other is an intracellular signaling module. The activation of modular CARs is based on two conditions, one is that the engineered T cell recognizes the antigen on the tumor cell surface through an antigen recognition module, the engineered T cell binds to the tumor cell but is not itself activated, and only when an external small molecule compound is present, the antigen recognition module and signaling module coupling can transmit an activation signal to kill the tumor cell (Wu CY, roybal KT, puchner EM, on J, lim wa. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor [ J ].2science.2015,350 (6258): aab 4077). However, the activation of the above-described modular CAR relies strictly on the addition of external small molecule compounds, and the appropriate timing of the application and deactivation of external small molecule compounds presents a great challenge to clinical applications.
Modern medical research has found that hypoxia is closely related to a number of diseases, such as arthritis, diabetic retinopathy, ischemic heart disease, stroke, etc., in particular solid tumors. Hypoxia is a common characteristic signal for many solid tumors because of insufficient blood supply and excessive proliferation of tumor cells due to abnormal vascular structures within the solid tumor, creating a relatively hypoxic tumor microenvironment, with oxygen levels within the tumor tissue often being less than 2% (Brown JM, wilson wr. Exploiding tumour hypoxia in Cancer treatment [ J ]. Nat Rev Cancer,2004, 4:437-447). Therefore, how to realize the clinical application of the modularized CAR in tumor treatment by using the difference of tumor microenvironment and normal tissue microenvironment and taking the difference as an influence factor to strictly control the activation drive of the modularized CAR, and the problems of inconvenience and inaccuracy caused by the activation of the modularized CAR driven by externally applying a small molecular compound in the prior art can be solved.
Disclosure of Invention
In view of the foregoing problems with the prior art, it is an object of the present invention to provide a conditionally controlled spliceable Chimeric Antigen Receptor (CAR) molecule and its use in the treatment of hypoxic diseases such as solid tumors, in particular in CAR-T cell therapy of solid tumors.
In the present invention, the description or definition of some terms is as follows:
the term "spliceable" refers to the fact that a protein molecule can be spliced back after it has been sheared.
The term "antigen recognition unit" refers to a protein molecule capable of specifically binding and recognizing a target antigen.
The term "signal transduction unit" refers to a protein molecule capable of transmitting a signal.
The term "antigen recognition domain" refers to a functional domain capable of specifically recognizing a target antigen, and generally includes Single chain antibodies (Single-chain fragment variable, scFv), single domain antibodies (Single domain antibody, sdAb), and extracellular ends of receptors, among others.
The term "transmembrane domain" refers to the region of a protein sequence that spans the cell membrane.
The term "costimulatory signal domain" refers to the domain in which different costimulatory molecules expressed on the surface of immune cells involved in adaptive immunity and their ligands bind to each other to generate costimulatory signals, typically costimulatory signal domains of costimulatory molecules selected from the group consisting of CD27, CD28, 4-1BB, OX40 and ICOS.
The term "N-terminal splice domain" refers to the N-terminal domain of an intein that can be spliced with the C-terminal splice domain.
The term "degrader" refers to a specific amino acid sequence that is recognized by proteases within a cell to mediate the clearance of degradation of a protein of interest.
The term "conditional signal response domain" refers to a specific amino acid sequence that can respond to specific conditional signals, including oxygen content, light and temperature, and regulate the degradation and enrichment of a protein of interest.
The term "C-terminal splice domain" refers to the C-terminal domain of an intein that can be spliced with the N-terminal splice domain.
The term "signaling domain" refers to a signaling domain of a T cell receptor comprising multiple immune receptor tyrosine activation motifs (Immunoreceptor tyrosine-based activation motif, ITAM), including cd3γ, cd3δ, cd3ε, and cd3ζ.
The term "hypoxic disease" refers to diseases, in particular solid tumors, in which the tissue oxygen content is less than 1%.
The invention aims at realizing the following technical scheme:
in a first aspect, the invention provides a conditionally controlled spliceable chimeric antigen receptor molecule comprising an antigen recognition unit and a signal transduction unit, wherein the antigen recognition unit comprises an antigen recognition domain, a transmembrane domain, a co-stimulatory signaling domain, an N-terminal splicing domain, and a degradant; the signal transduction unit comprises a conditional signal response domain, a C-terminal splice domain, and a signaling domain.
The conditionally controlled splice chimeric antigen receptor molecules provided herein are capable of effecting splicing of antigen recognition units and signal transduction units in response to specific conditional signals, such as hypoxia. Wherein the antigen recognition unit can spontaneously/induce degradation, reduce retention in normal tissues, and the signaling unit can respond to specific condition signals, such as hypoxia, and has the characteristics of low expression in normal tissue environments and enrichment in tumor microenvironment.
In the chimeric antigen receptor molecules according to the invention, the antigen to which the antigen recognition domain binds may be selected from one or more of the CD47, AXL, EGFR, CD7, CD24, FAP, CD147, HER2, ROR1, ROR2, CD133, ephA2, CD171, CEA, epCAM, TAG, IL-13 ra, egfrvlll, GD2, fra, PSCA, PSMA, GPC3, CAIX, claudin18.2, VEGFR2, PD-L1, MSLN, MUC1, c-Met, B7-H3 or TROP2 antigens. Preferably, the antigen to which the antigen recognition domain binds is the CD47 antigen.
In the chimeric antigen receptor molecules according to the invention, the transmembrane domain may be selected from one or more of the cd3ζ, CD4, CD8, CD28 or CD137/4-1BB transmembrane domains. Preferably, the transmembrane domain is a CD28 transmembrane domain.
In the chimeric antigen receptor molecules according to the invention, the costimulatory domain in the antigen recognition unit can be selected from one or more of the CD2, CD27, CD28, CD40, OX40, CD137/4-1BB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11 or Dap10 costimulatory domains. Preferably, the co-stimulatory domain in the antigen recognition unit is selected from one or more of the CD28 or CD137/4-1BB co-stimulatory domains.
In the chimeric antigen receptor molecules according to the invention, the N-terminal splice domain may be selected from one or more of the protein introns or SpyTag/SpyCatcher self-assemblies. Preferably, the N-terminal splice domain is the protein intron Int N 。
In the chimeric antigen receptor molecule according to the present invention, the degradative molecule may be selected from one or more of dihydrofolate reductase (DHFR), estrogen Receptor (ER), salmonella type III secretory system effector protein (SopE), phytohormone-induced protein degradative molecule, unstable domain (AD), or photosensitive protein degradative molecule. Preferably, the degradant is selected from one or more of the estrogen receptor or salmonella type III secretory system effector proteins. More preferably, the degradation element is a mutant estrogen receptor (ERm).
In the chimeric antigen receptor molecules according to the invention, the condition signal response domain may be selected from one or more of an oxygen dependent degradation domain (ODD), a temperature sensitive domain, a pH sensitive domain, a photoactive domain or an inflammatory factor response domain. Preferably, the conditional signal response domain is an oxygen dependent degradation domain.
In the chimeric antigen receptor molecules according to the invention, the C-terminal splice domain may be selected from one or more of the protein introns or SpyTag/SpyCatcher self-assemblies. Preferably, the C-terminal splice domain is the protein intron Int C 。
In the chimeric antigen receptor molecules according to the invention, the signaling domain may be selected from one or more of CD3 ζ, fcγriii, fcεri, fc receptor signaling domain, or an immunoreceptor tyrosine-activating motif (ITAM) -bearing signaling molecule. Preferably, the signaling domain is a CD3 ζ signaling domain.
In the chimeric antigen receptor molecule according to the invention, preferably, the signal transduction unit further comprises a costimulatory signal domain. More preferably, the costimulatory signaling domain in the signaling unit may be selected from one or more of the CD2, CD27, CD28, CD40, OX40, CD137/4-1BB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11 or Dap10 costimulatory signaling domains.
According to a preferred embodiment of the invention, the antigen recognition unit has the amino acid sequence shown in SEQ NO:1 or SEQ NO: 2.
According to a preferred embodiment of the invention, the amino acid sequence of the signal transduction unit is set forth in SEQ NO: 3.
In a second aspect, the present invention provides a nucleic acid molecule encoding a chimeric antigen receptor molecule according to the invention as described above.
In the nucleic acid molecule according to the invention, preferably the nucleotide sequence encoding the signal transduction unit further comprises a nucleotide sequence encoding a conditional signal response element. Preferably, the condition signal responsive element is selected from one or more of an Hypoxia Responsive Element (HRE), a temperature sensitive element, a pH sensitive element, a light sensitive element or an inflammatory factor responsive element. More preferably, the condition signal responsive element is a Hypoxia Responsive Element (HRE). The signal transduction unit can further enhance the response ability to a condition signal at the nucleic acid level under the control of a nucleotide sequence as a hypoxia response element.
According to a preferred embodiment of the invention, the nucleotide sequence encoding said signal transduction unit is set forth in SEQ NO: 4.
According to a preferred embodiment of the invention, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ NO:5 or 6.
In a third aspect, the present invention provides a vector comprising a nucleic acid molecule according to the invention as described above.
The vector according to the invention may be selected from one or more of a plasmid, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a herpes simplex viral vector, a forest encephalitis viral vector, a polio viral vector, a newcastle disease viral vector or a transposon. Preferably, the vector is a lentiviral vector.
In a fourth aspect, the present invention provides a genetically engineered host cell comprising an exogenous nucleic acid molecule according to the invention as described above, or comprising a vector according to the invention as described above, integrated in its chromosome.
The host cell according to the invention may be selected from one or more of an isolated human cell or a genetically engineered immune cell.
Preferably, the isolated human-derived cells are selected from one or more of embryonic stem cells, umbilical cord blood-derived stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, T cells, NK cells, NKT cells, or macrophages.
Preferably, the genetically engineered immune cells are selected from one or more of genetically engineered T cells, NK cells, NKT cells or macrophages. More preferably, the genetically engineered immune cell is a genetically engineered T cell.
According to a preferred embodiment of the invention, the genetically engineered immune cell is selected from the group consisting of chimeric antigen receptor T cells (CAR-T cells), chimeric antigen receptor NK cells (CAR-NK cells), chimeric antigen receptor NKT cells (CAR-NKT cells), chimeric antigen receptor macrophagesOr one or more of the T cell receptor T cells (TCR-T cells). According to a more preferred embodiment of the invention, the genetically engineered immune cells are chimeric antigen receptor T cells.
A host cell genetically engineered according to the invention is capable of driving splicing of a modular CAR in an anoxic environment by transduction of the nucleic acid molecule according to the invention described above or transfection of the vector according to the invention described above.
The invention also provides a preparation method of the genetically engineered host cell, which comprises the step of introducing the nucleic acid molecule and/or the vector into the host cell.
In the preparation method according to the present invention, the introduction may be transfection or transduction.
In a fifth aspect, the present invention provides the use of a chimeric antigen receptor molecule, nucleic acid molecule, vector or genetically engineered host cell as described above for the preparation of a cellular immunotherapeutic agent according to the invention.
Accordingly, the present invention provides a method of cellular immunotherapy comprising:
introducing the above-described nucleic acid molecule and/or vector according to the invention into a host cell, administering to a subject in need thereof a therapeutically effective amount of said host cell, or
Administering to a subject in need thereof a therapeutically effective amount of a genetically engineered host cell according to the invention as described above.
Preferably, the introduction is simultaneous, sequential or sequential.
Preferably, in the above use or method according to the invention, the cellular immunotherapeutic agent or method is for the treatment of a hypoxic disease. Preferably, the hypoxic disease is cancer. More preferably, the cancer is a solid tumor, such as one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, stomach cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, or brain cancer.
The inventor finds that the activation of the modularized CAR driven by the intrinsic characteristic signals of the tumor microenvironment has more clinical application value. The tumor hypoxia microenvironment signal is hopeful to be used for developing new technology for treating hypoxia diseases such as solid tumors, and the specificity of tumor treatment can be improved by activating the modularized CAR through the hypoxia microenvironment signal, so that the damage to normal tissues is avoided or reduced.
The conditionally controlled spliceable chimeric antigen receptor molecules provided by the invention comprise an antigen recognition unit and a signal transduction unit, which respond to specific conditional signals (such as hypoxia) and effect splicing of the antigen recognition unit and the signal transduction unit, and exhibit the following three advantages:
1) The antigen recognition unit can spontaneously/induce degradation, so that retention in normal tissues is reduced, and damage to the normal tissues is reduced;
2) The signal transmission unit can respond to specific condition signals (such as hypoxia), and has the characteristics of low expression in normal tissue environment and enrichment in tumor microenvironment;
3) The modular CAR splicing controlled by the condition realizes the specific activation in the tumor microenvironment, and effectively reduces the on-target off-tumor toxic and side effects caused by the targeting of CAR-T cells to non-tumor tissues.
It is understood that within the scope of the present invention, the above technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. Is limited to space limitations and will not be described in detail herein.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows a conventional CD47 CAR (CD 47-28BBz, FIG. 1 a), a hypoxia regulated CD47 CAR-ODD (CD 47-28BBz-ODD, FIG. 1 b) fused to an oxygen dependent degradation domain ODD, an antigen recognition unit 1 (CD 47-28 BB-Int) N SopE, FIG. 1 c), antigen recognition unit 2 (CD 47-28 BB-Int) N ERm, FIG. 1 d), hypoxia regulated signal transduction unit (ODD-Int C -CD3z, FIG. 1 e), two-in-one conditional control (hypoxia regulated) alternative splicing system 1 (CD 47-28 BB-Int) N -SopE-HRE-ODD-Int C -CD3z, FIG. 1 f) and two-in-one conditional control (hypoxia regulated) alternative splicing System 2 (CD 47-28 BB-Int) N -ERm-HRE-ODD-Int C -CD3z, figure 1 g).
FIGS. 2a-c show antigen recognition unit 1 (CD 47-28 BB-Int) N SopE) and antigen recognition unit 2 (CD 47-28 BB-Int) N ERm) spontaneous degradation. Wherein fig. 2a shows the structure of the antigen recognition unit and its constituent parts; FIG. 2b is a flow cytometry detection result on 293T cell membrane of CD47 CAR fused with oxygen-dependent degradation domain ODD alone, antigen recognition unit 1 fused with degradation subunit SopE, and antigen recognition unit 2 fused with degradation subunit ERm; fig. 2c is a statistical result of the flow cytometry detection data of fig. 2b, the spontaneous degradation capacity of the degradation seeds SopE and ERm is obviously better than that of the oxygen-dependent degradation domain ODD, and the degradation capacity of ERm is optimal.
FIGS. 3a-d show antigen recognition unit 1 (CD 47-28 BB-Int) N SopE) and antigen recognition unit 2 (CD 47-28 BB-Int) N ERm) splicing Activity. Wherein, FIG. 3a shows the structure of the degradative fusion CAR and hypoxia regulatory signaling unit ODD-IntC-CD3z and its composition; FIG. 3b shows an integrated CAR and the hypoxia regulated signal transduction unit ODD-IntC-CD3z engineering 293T cells and the integrated degradation seed fusion CAR and hypoxia regulated signal transduction unit ODD-IntC-CD3z engineering 293T cells are subjected to CAR expression level under the hypoxia condition, wherein Blank is a flow detection result of the negative 293T cells, and Mock represents the non-hypoxia regulated signal transduction unit ODD-IntC-CD3z engineering 293T cells, namely empty virus transduction engineering 293T cells; FIG. 3c is fold induction of CAR expression levels after splicing under hypoxic conditions; FIG. 3d shows that three genetically engineered 293T cells simultaneously integrated with the hypoxia regulated signaling unit ODD-IntC-CD3z were placed in normoxic (21% O) 2 ) And anaerobic conditions (1%O) 2 ) Culturing for 24h, and collecting the results of Western blotting detection of the cells after culturing.
FIG. 4 shows transduction of CD47 CAR (CD 47-28BBz, where z represents the CD3 zeta signaling domain), antigen recognition unit 1 (CD 47-28 BB-Int) N SopE), two-in-one conditional control (hypoxia regulated) alternative splicing System 1 (CD 47-28 BB-Int) N -SopE-HRE-ODD-Int C -CD3 z), antigen recognition unit 2 (CD 47-28 BB-Int) N ERm) and two-in-one conditional control (hypoxia regulated) alternative splicing System 2 (CD 47-28 BB-Int) N -ERm-HRE-ODD-Int C -CD3 z) CAR molecular flow cytometry detection results on engineered T cell membranes. Fig. 4b shows statistics of the streaming detection data of fig. 4 a.
FIG. 5 shows transduction of CD47 CAR (CD 47-28 BBz), antigen recognition unit 1 (CD 47-28 BB-Int) N SopE), two-in-one conditional control (hypoxia regulated) alternative splicing System 1 (CD 47-28 BB-Int) N -SopE-HRE-ODD-Int C -CD3 z), antigen recognition unit 2 (CD 47-28 BB-Int) N ERm) and two-in-one conditional control (hypoxia regulated) alternative splicing System 2 (CD 47-28 BB-Int) N -ERm-HRE-ODD-Int C Normoxic and hypoxia-dependent cell killing results of engineered T cells of CD3 z). The target cell of fig. 5a is the ovarian cancer tumor cell line SKOV3; the target cell of FIG. 5b is the lung cancer tumor cell line NCI-H292.
FIG. 6 shows a two-in-one condition-controlled (hypoxia regulated) alternative splicing system 1 (CD 47-28 BB-Int) N -SopE-HRE-ODD-Int C -CD3 z) and two-in-one conditional control (hypoxia regulated) alternative splicing system 2 (CD 47-28 BB-Int) N -ERm-HRE-ODD-Int C -CD3 z) in vivo anti-tumor activity of engineered T cells. Fig. 6a is a flow chart of the experiment, and fig. 6b is a statistical result of tumor size 30 days after infusion of the engineered T cells.
Detailed Description
The advantages and features of the present invention will become more apparent from the following description of the embodiments. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention.
The experimental methods in the following examples, unless otherwise specified, are all routine in the art. The experimental materials used in the following examples, unless otherwise specified, were all conventional biochemical reagents, purchased from the sales company, wherein:
DMEM medium, RPMI1640 medium, were all purchased from Corning corporation; lymphocyte culture medium X-VIVO 15 was purchased from Lonza corporation.
The specific preparation method of the T cell growth medium is shown in Chinese patent application CN201910163391.1, and the T cell growth medium consists of a basic medium and cytokines, wherein the basic medium is lymphocyte culture medium X-VIVO 15, and the cytokines of 5ng/mL IL-7, 10ng/mL IL-15 or 30ng/mL IL-21 are additionally added. Among them, cytokines IL-7 and IL-15 were purchased from R & D, and IL-21 was purchased from offshore protein technologies Inc.
Fetal bovine serum was purchased from BI company.
The TurboFect transfection kit was purchased from Thermo Fisher Scientific company.
Lenti-X lentiviral concentrate reagent was purchased from Takara.
The gene synthesis was performed by Shanghai JieRui bioengineering Co.
The blank lentivirus expression plasmids (pXW-EF 1 alpha-MCS-P2A-EGFP and pXW-EF1 alpha-BFP-MCS 1-HRE-MCS 2) are derived from commercial plasmids ABpCCLsin-EF1 alpha-MCS purchased from Kang Lin biotechnology (Hangzhou) limited, and the green fluorescent protein gene and the blue fluorescent protein gene are recombined on the commercial plasmids respectively, so that the blank lentivirus expression plasmid is obtained, and the packaging plasmid psPAX2 and the envelope plasmid PMD2.G are purchased from Addgene.
Stable 3 chemically competent cells were purchased from Shanghai Biotechnology, inc.
The endotoxin-free plasmid miniprep kit and the endotoxin-free plasmid midprep kit were purchased from OMEGA and Macherey Nagel, respectively.
Luciferase substrates were purchased from Promega Biotechnology Co.
293T cells, A549 lung cancer cells, SKOV3 ovarian cancer cells, and NCI-H292 lung cancer cells were purchased from ATCC in the United states. The SKOV3-luc and NCI-H292-luc with stable integration of firefly luciferase gene were obtained by transduction of SKOV3 ovarian cancer cells and NCI-H292 lung cancer cells with lentivirus carrying firefly luciferase gene.
Severe combined immunodeficiency mice (B-NDG) were purchased from the biotechnological company, inc.
EXAMPLE 1 conditional control of the composition of a chimeric antigen receptor molecule that can be spliced
The amino acid sequence of the antigen recognition unit is shown in SEQ NO:1 or SEQ NO:2, SEQ NO:1 or SEQ NO:2 consists of the sequences of the CD47 antigen recognition domain, the CD28 transmembrane domain, the CD28 and 4-1BB costimulatory signal domain, the N-terminal splice domain and the degrader, respectively. The amino acid sequences of specific CD47 antigen recognition domains, CD28 transmembrane domains, CD28 and 4-1BB costimulatory signal domains, N-terminal splice domains and degradants are shown in table 1.
TABLE 1 amino acid sequence of antigen recognition units
The amino acid sequence of the signal transduction unit is shown in SEQ NO:3, SEQ NO:3 consists of sequences of hypoxia condition signal response domain (oxygen dependent degradation domain ODD), C-terminal splice domain, CD3 zeta signaling domain. The amino acid sequences of specific hypoxia condition signal response domains (oxygen dependent degradation domain ODD), C-terminal splice domain and CD3 zeta signaling domain are shown in table 2.
TABLE 2 amino acid sequences of Signal transduction units
The nucleotide sequence of the signal transduction unit containing the hypoxia response element is shown as SEQ NO:4, SEQ NO:4 consists of sequences of a hypoxia condition signal response domain (oxygen dependent degradation domain ODD), a C-terminal splice domain, a CD3 zeta signaling domain and a hypoxia response element. The nucleotide sequences of specific hypoxia condition signal response domains (oxygen dependent degradation domains ODDs), C-terminal splice domains, CD3 zeta signaling domains and hypoxia response elements are shown in table 3.
TABLE 3 nucleotide sequences of Signal transduction units
The nucleotide sequence of the chimeric antigen receptor molecule capable of splicing is controlled by the condition as shown in SEQ NO:5 or SEQ NO:6, SEQ NO:5 consists of a nucleic acid coding sequence for the CD47 antigen recognition domain, a nucleic acid coding sequence for the CD28 transmembrane domain, a nucleic acid coding sequence for the CD28 and 4-1BB co-stimulatory signal domain, a nucleic acid coding sequence for the N-terminal splice domain, a nucleic acid coding sequence for the degradation element SopE, a Hypoxia Response Element (HRE), a nucleic acid coding sequence for the hypoxia condition signal response domain (oxygen dependent degradation domain ODD), a nucleic acid coding sequence for the C-terminal splice domain and a nucleic acid coding sequence for the CD3 zeta signaling domain; SEQ NO:6 consists of the nucleic acid coding sequence of the CD47 antigen recognition domain, the nucleic acid coding sequence of the CD28 transmembrane domain, the nucleic acid coding sequence of the CD28 and 4-1BB co-stimulatory signaling domains, the nucleic acid coding sequence of the N-terminal splice domain, the nucleic acid coding sequence of the degrader ERm, the Hypoxia Response Element (HRE), the nucleic acid coding sequence of the hypoxia condition signal response domain (oxygen dependent degradation domain ODD), the nucleic acid coding sequence of the C-terminal splice domain and the nucleic acid coding sequence of the CD3 zeta signaling domain. The nucleic acid coding sequence for a specific CD47 antigen recognition domain, the nucleic acid coding sequence for a CD28 transmembrane domain, the nucleic acid coding sequence for the CD28 and 4-1BB co-stimulatory signal domains, the nucleic acid coding sequence for the N-terminal splice domain, the nucleic acid coding sequence for the degradation element SopE/ERm, the Hypoxia Response Element (HRE), the nucleic acid coding sequence for the hypoxia condition signal response domain (oxygen dependent degradation domain ODD), the nucleic acid coding sequence for the C-terminal splice domain and the nucleic acid coding sequence for the CD3 zeta signaling domain are shown in table 4.
TABLE 4 nucleotide sequences of chimeric antigen receptor molecules that conditionally control splicing
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EXAMPLE 2 construction of lentiviral expression plasmids
SEQ NO was synthesized by Shanghai JieRui bioengineering Co.Ltd: 7-9, and cloning to a blank lentiviral expression plasmid, respectively, to obtain:
recombination with pXW-EF1 alpha-MCS-P2A-EGFP yields a polypeptide carrying the nucleic acid sequence SEQ NO:7 pXW-EF1 a-CD 47-28BBz-P2A-EGFP recombinant plasmid, i.e. a lentiviral expression plasmid of a conventional CD47CAR (abbreviated as pXW-CD47 CAR) in which the CD47-28BBz gene is recombined. Wherein, SEQ NO:7 encodes a CD47 antigen recognition domain, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain (see tables 1 and 2 for specific sequences of each component).
Recombination with pXW-EF1 alpha-MCS-P2A-EGFP yields a polypeptide carrying the nucleic acid sequence SEQ NO:8 pXW-EF1 alpha-CD 47-28BBz-ODD-P2A-EGFP recombinant plasmid, namely a lentiviral expression plasmid fusing oxygen dependent degradation domain ODD for hypoxia regulation of CD47 CAR-ODD (abbreviated as pXW-CD47 CAR-ODD, recombinant CD47-28BBz-ODD gene). Wherein, SEQ NO:8 encodes a CD47 antigen recognition domain, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, a 4-1BB costimulatory signaling domain, a CD3 zeta signaling domain, and a hypoxia condition signal response domain (i.e., an oxygen dependent degradation domain ODD) (see tables 1 and 2 for specific sequences of the components).
Recombination with pXW-EF1 alpha-MCS-P2A-EGFP yields a polypeptide carrying the nucleic acid sequence SEQ NO:9 pXW-EF1 alpha-CD 47-28BB-Int N The SopE-P2A-EGFP recombinant plasmid, i.e.the lentiviral expression plasmid of antigen recognition unit 1 (abbreviated as pXW-CD47 CAR-Int N SopE, recombinant CD47-28BB-Int N The SopE gene). Wherein, SEQ NO:9 encodes a CD47 antigen recognition domain, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, a 4-1BB costimulatory signaling domain, an N-terminal splice domain, and a degradation element SopE (see tables 1 and 2 for specific sequences of the components).
Recombination with pXW-EF1 alpha-MCS-P2A-EGFP yields a polypeptide carrying the nucleic acid sequence SEQ NO:10 pXW-EF1 alpha-CD 47-28BB-Int N -ERm-P2A-EGFP recombinant plasmid, i.e.lentiviral expression plasmid of antigen recognition unit 2 (abbreviated as pXW-CD47 CAR-Int N ERm, recombinant CD47-28BB-Int N ERm gene). Wherein, SEQ NO:10 encodes a CD47 antigen recognition domain, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, a 4-1BB costimulatory signaling domain, an N-terminal splice domain, and a degradant ERm (see tables 1 and 2 for specific sequences of each component).
Recombination with pXW-EF1 alpha-BFP-MCS 1-HRE-MCS2 yields a polypeptide carrying the nucleic acid sequence SEQ NO:11 pXW-EF1 alpha-BFP-HRE-ODD-Int C CD3z, the hypoxia regulated Signal transduction Unit ODD-Int C- Lentiviral expression plasmid of CD3z (abbreviated as pXW-ODD-Int C CD3z, recombinant ODD-Int C -CD3z gene). Wherein, SEQ NO:11 encodes a hypoxia condition signal response domain (oxygen dependent degradation structure ODD), a C-terminal splice domain and a CD3 zeta signaling domain (see tables 1 and 2 for specific sequences of the components).
Recombination with pXW-EF1 alpha-BFP-MCS 1-HRE-MCS2 yields a recombinant vector carrying the nucleic acid sequence SEQ NO:9 and SEQ NO:11 pXW-EF1 alpha-BFP-CD 47-28BB-Int N -SopE-HRE-ODD-Int C- CD3z (abbreviated pXW-CD47 CAR-Int N -SopE-HRE-ODD-Int C -CD3 z) (see tables 1, 2 and 3 for specific sequences of the individual components).
Recombination with pXW-EF1 alpha-BFP-MCS 1-HRE-MCS2 yields a recombinant vector carrying the nucleic acid sequence SEQ NO:10 and SEQ NO:11 pXW-EF1 alpha-BFP-CD 47-28BB-Int N -ERm-HRE-ODD-Int C CD3z (abbreviated pXW-CD47 CAR-Int N -ERm-HRE-ODD-Int C- CD3 z) (see tables 1, 2 and 3 for specific sequences of the individual components).
The respective recombinant plasmid maps are shown in FIG. 1.
EXAMPLE 3 packaging, concentration and titre determination of lentiviruses
1.1 packaging of lentiviruses
293T cell treatment: 24h before transfection, 293T cells in logarithmic growth phase were collected and inoculated into 10cm cell culture dishes (6-8X10) 6 Individual cells) were grown in complete DMEM medium containing 10mL, placed at 37 ℃, 5% co 2 Culturing in a cell culture box for 18-24h, wherein the cell density reaches 70-90%, and plasmid transfection can be performed.
Transfection of 293T cells: 1mL of basic DMEM culture medium is added into a 15mL centrifuge tube, and lentivirus expression plasmids are added according to the mass ratio: packaging plasmid (psPAX 2): envelope plasmid (pmd 2. G) =1: 3: 1A transfection mixture was prepared with 7 plasmids prepared in example 2, the total amount of plasmids totaling 15. Mu.g/dish. Dividing intoThe plasmid amount (μg): transfection reagent (μl) =1: 2, 30 mu L of TurboFect transfection reagent is added in proportion, incubated for 15-20min at room temperature, added into a culture dish paved with 293T cells, and placed at 37 ℃ and 5% CO 2 After 48 hours of continuous culture in the cell incubator, the virus supernatant was collected, centrifuged at 1000 Xg at 4℃for 10min, the pellet at the bottom of the tube was discarded and the virus supernatant was collected.
1.2 concentration of lentiviruses
Further filtering the virus supernatant collected by centrifugation by using a 0.45 μm filter, adding a Lenti-X lentiviral concentration reagent with the volume of 1/3 of the virus supernatant, mixing the mixture for a plurality of times in a reverse manner, incubating the mixture at 4 ℃ overnight, centrifuging the mixture at 2000 Xg for 45min at 4 ℃, and obtaining the concentrated virus particles by visible white precipitation at the bottom of the centrifuge tube. The supernatant was carefully discarded, and the white pellet was resuspended in 1/20 of the volume of blank RPMI1640 medium from proviral supernatant, split at 250. Mu.L and frozen at-80℃for use.
1.3 lentiviral titre assay
Jurkat T cells were plated at 1X 10 5 The lentiviral concentrate collected was diluted 10-fold in increments by inoculating the cells/well onto a 96-well U-bottom plate. 100 mu L of virus diluent is added into a corresponding hole, an infection-promoting reagent protamine sulfate is added, the concentration is adjusted to 10 mu g/mL,1000 Xg is carried out, centrifugal infection is carried out for 90min at 32 ℃, fresh RPMI1640 complete medium is replaced after overnight culture, the culture is continued for 48h, and the proportion of fluorescent positive cells is detected by a flow cytometer. Viral titers were calculated using the following formula:
virus titer (TU/mL) =1×10 5 X fluorescence positive cell fraction/100 x 1000 x corresponding dilution.
EXAMPLE 4 preparation of genetically engineered T cells
Example 3 the following lentiviral vectors were obtained by concentration:
LV-EF1 alpha-CD 47-28BBz-P2A-EGFP (obtained from pXW-EF1 alpha-CD 47-28 BBz-P2A-EGFP);
LV-EF1 alpha-CD 47-28BBz-ODD-P2A-EGFP (obtained from pXW-EF1 alpha-CD 47-28 BBz-ODD-P2A-EGFP);
LV-EF1α-CD47-28BB-Int N SopE-P2A-EGFP (from pXW-EF 1. Alpha. -CD47-28 BB-Int) N -SopE-P2A-EGFP);
LV-EF1α-CD47-28BB-Int N ERm-P2A-EGFP (from pXW-EF 1. Alpha. -CD47-28 BB-Int) N -ERm-P2A-EGFP);
LV-EF1 alpha-BFP-MCS 1-HRE-MCS2 (obtained from pXW-EF1 alpha-BFP-MCS 1-HRE-MCS 2);
LV-EF1α-BFP-HRE-ODD-Int C CD3z (pXW-EF 1. Alpha. -BFP-HRE-ODD-Int C -CD3z acquisition);
LV-EF1α-BFP-CD47-28BB-Int N -SopE-HRE-ODD-Int C CD3z (pXW-EF 1. Alpha. -BFP-CD47-28BB-Int N -SopE-HRE-ODD-Int C -CD3z acquisition);
LV-EF1α-BFP-CD47-28BB-Int N -ERm-HRE-ODD-Int C CD3z (pXW-EF 1. Alpha. -BFP-CD47-28BB-Int N -ERm-HRE-ODD-Int C CD3z acquisition).
Each of the above lentiviral vectors was added to the same bed at MOI=5 by 1×10 6 The method comprises the steps of adding an infection-promoting reagent protamine sulfate into a 48-well flat bottom plate of peripheral blood mononuclear cells which are activated for 3 days by using an equal number of immunomagnetic beads in advance, adjusting the working concentration to 10 mug/mL, centrifugally infecting for 90min at the temperature of 1000 Xg and 32 ℃, and continuously culturing by replacing fresh T cell growth medium after overnight culturing.
Fresh T cell growth medium was added every 2-3 days and cell density was adjusted to 0.5-2X 10 6 Individual cells/mL. And removing immunomagnetic beads of activated T cells 6-7 days after infection, continuously culturing and amplifying genetically engineered T cells, and carrying out subsequent functional experiments after the cells are at rest (9-14 days after the magnetic beads are removed).
Example 5 detection of expression level of a degradative fusion CAR molecule
To verify the spontaneous degradability of the degradants of the invention, plasmid transfection experiments were performed in 293T cells in this example.
293T cells were plated at 5X 10 4 Individual cells/wells were plated into 48-well plates and transfected after the next day of cell attachment. 0.5 μg of recombinant expression plasmid pXW-CD47CAR, pXW-CD47CAR-ODD, pXW-CD47 CAR-Int were transfected separately N SopE or pXW-CD47 CAR-Int N ERm, in plasmid quantity (. Mu.g): transfection reagent(μl) =1: 2, adding a TurboFect transfection reagent in proportion, incubating for 15-20min at room temperature, adding into a cell culture plate, and placing at 37deg.C with 5% CO 2 After culturing in a cell culture incubator for 48 hours, the expression of the CAR molecules on the cell membrane is detected in a flow assay. The results are shown in FIG. 2.
FIG. 2a shows the structure of a fusion CAR of the degradants SopE or ERm placed at the C-terminal, N-terminal splice domain Int N The signal deleted CAR molecule and the degradant are linked.
Figure 2b shows the results of flow cytometry detection of the expression levels of positive control CD47CAR (CD 47-28 BBz), oxygen dependent degradation domain ODD fusion CD47CAR (CD 47 CAR-ODD), and two degradation sub fusion CARs. The results indicate that although there was little difference in CAR positive rate between groups, the level of expression of the degradant SopE or ERm fusion CARs was significantly lower than the positive control CD47CAR, as well as lower than the CD47 CAR-ODD.
The statistics of figure 2c further demonstrate that the expression level of the degradants SopE or ERm fusion CAR is only 10% or 7% of the positive control CD47CAR, the spontaneous degradation rate is as high as 90% or 93%, and is also superior to the 54% degradation rate of the oxygen dependent degradation domain ODD fusion CD47 CAR.
EXAMPLE 6 splicing Activity of the conditionally controlled alternative splicing System
The lentiviral expression plasmid constructed in example 2 and the lentiviral vector preparation method of example 3 were used to obtain the corresponding lentiviral vector.
This example performed lentiviral vector transduction experiments in 293T cells and tested splice activity of a conditionally controlled alternative splicing system. The recombinant lentiviral vectors and empty viral vectors (LV-) were each added to a 1X 10 spread with MOI=5 alone or in combination 6 The 6-well flat bottom plate of each 293T cell is added with polycoagulamine (Polybrene) which is a pro-infective agent and the working concentration is adjusted to 10 mug/mL, after overnight culture, fresh cell growth culture medium is changed for continuous culture to obtain stable integration 1) EF1 alpha-CD 47-28BBz-P2A-EGFP, 2) EF1 alpha-CD 47-28BB-Int N -SopE-P2A-EGFP、3)EF1α-CD47-28BB-Int N -ERm-P2A-EGFP, 4) EF1 alpha-CD 47-28BBz-P2A-EGFP and hypoxia regulated signaling unit EF1 alpha-BFP-HRE-ODD-Int C- CD3z、5)EF1α-CD47-28BB-Int N SopE-P2A-EGFP and hypoxia regulated signaling unit EF1 alpha-BFP-HRE-ODD-Int C- CD3z、6)EF1α-CD47-28BB-Int N -ERm-P2A-EGFP and hypoxia regulated signaling unit EF1 alpha-BFP-HRE-ODD-Int C- CD3z, 7) EF1 alpha-CD 47-28BBz-P2A-EGFP and EF1 alpha-BFP-MCS 1-HRE-MCS2 (Mock), 8) EF1 alpha-CD 47-28BB-Int N -SopE-P2A-EGFP and EF1 alpha-BFP-MCS 1-HRE-MCS2 (Mock), 9) EF1 alpha-CD 47-28BB-Int N 9 different genetically engineered 293T cells of-ERm-P2A-EGFP and EF1 alpha-BFP-MCS 1-HRE-MCS2 (Mock). The 9 genetically engineered 293T cells were placed under hypoxic conditions (1%O) 2 ) After culturing for 24 hours, collecting cells after culturing, eluting with FACS buffer for 1 time, adding 2 mug/mL of flow antibody PE-anti-DYKDDDK, incubating at room temperature for 20 minutes in dark, eluting with FACS buffer for 2 times after finishing, re-suspending with 300 mug FACS buffer, and detecting the expression of CAR molecules by using a flow cytometer. In addition, stable integration 1) EF1 alpha-CD 47-28BBz-P2A-EGFP, 2) EF1 alpha-CD 47-28BB-Int N -SopE-P2A-EGFP、3)EF1α-CD47-28BB-Int N -ERm-P2A-EGFP, 4) EF1 alpha-CD 47-28BBz-P2A-EGFP and hypoxia regulated signaling unit EF1 alpha-BFP-HRE-ODD-Int C- CD3z、5)EF1α-CD47-28BB-Int N SopE-P2A-EGFP and hypoxia regulated signaling unit EF1 alpha-BFP-HRE-ODD-Int C- CD3z、6)EF1α-CD47-28BB-Int N -ERm-P2A-EGFP and hypoxia regulated signaling unit EF1 alpha-BFP-HRE-ODD-Int C- CD3z 6 genetically engineered 293T cells were placed in normoxic (21% O) 2 ) And anaerobic conditions (1%O) 2 ) Culturing for 24h, and collecting cells for western blotting detection after culturing. The results are shown in FIG. 3.
FIG. 3a shows degradation of the fusion CAR and hypoxia regulated signaling unit ODD-Int C- CD3z structure and its composition, ODD-Int C- CD3z is encoded by the oxygen dependent degradation domain ODD, C-terminal splice domain Int C And signaling domain CD3 z.
FIG. 3b shows the results of a flow assay to integrate the degradation sub-fusion CAR with the hypoxia regulated signaling unit ODD-Int C- CAR expression levels of engineered cells of CD3z under hypoxic conditions were obtainedFurther promotes, prompts the signal transmission unit ODD-Int of hypoxia regulation and control C- CD3z is enriched under hypoxic conditions and effects splicing of the degradative fusion CAR, releasing the degradative molecule.
Figure 3c is fold induction of CAR expression levels after splicing, the degradation seed SopE or ERm fusion CAR fold induction was 4 or 5 fold, respectively, while the control CD47 CAR expression levels were unchanged.
The Western Blotting results of fig. 3d demonstrate that the successful splicing of the hypoxia-reduced fusion CAR and signaling unit is up to 100% efficient, hypoxia (1%O 2 ) Complete splicing is achieved and a single band of smaller molecular weight than the unspliced molecule is obtained.
EXAMPLE 7 conditional controlled alternative splicing System Regulation of chimeric antigen receptor expression
Preparation of stable integration of EF1 alpha-CD 47-28BBz-P2A-EGFP, EF1 alpha-CD 47-28BB-Int by the lentiviral expression plasmid constructed in example 2, the preparation method of lentiviral vector of example 3 and the preparation method of genetically engineered T cells described in example 4 N -SopE-P2A-EGFP、EF1α-BFP-CD47-28BB-Int N -SopE-HRE-ODD-Int C- CD3z、EF1α-CD47-28BB-Int N -ERm-P2A-EGFP or EF1 alpha-BFP-CD 47-28BB-Int N -ERm-HRE-ODD-Int C 5 engineered T cells of the CD3z gene. The above 5 genetically engineered T cells were placed in normoxic (21% O) 2 ) Or anoxic conditions (1%O) 2 ) After incubation for 24h, cells were collected after the end and eluted 1 pass with FACS buffer, 2 μg/mL of flow antibody PE-anti-dykdddk was added, incubated at room temperature for 20min protected from light, 2 passes with FACS buffer after the end, resuspended in 300 μl FACS buffer, and expression of HER2 CAR molecules was detected using a flow cytometer. The results are shown in FIG. 4.
FIG. 4a shows that positive control CD47 CAR-T cells (CD 47-28 BBz) both under normoxic and anoxic conditions highly express the CD47 CAR molecule, while the CAR molecules regulated by the conditionally controlled spliceable system are expressed low in normoxic environments, while expression is induced in anoxic environments, in particular stable integration of EF 1. Alpha. -BFP-CD47-28BB-Int N -SopE-HRE-ODD-Int C- Engineered T cells of CD3 z.
The statistics of fig. 4b demonstrate that the positive rate of the degradation son SopE or ERm fusion CAR under normoxic conditions is 46% or 24% of the positive control, respectively, the expression level is even lower to 4% or 2% of the control, and the induction fold is significantly up-regulated under anoxic conditions, 9 or 3 fold, respectively, suggesting better activity of the spliceable system based on conditional control of the degradation son SopE.
EXAMPLE 8 Condition-controlled alternative splicing System Regulation of tumor cell killing of chimeric antigen receptor T cells
Tumor cell killing efficiency was assessed by Luciferase-based cell killing assay (Luciferase-based cytotoxicity assay). First, 1×10 will be 4 Inoculating SKOV3-Luc (firefly luciferase gene modified human ovarian cancer cells) or NCI-H292-Luc (firefly luciferase gene modified human lung cancer cells) on 96-well flat-bottomed blackboard with 100 μl of culture medium per well, placing at 37deg.C and 5% CO 2 The cells were cultured in an incubator for 18h. The following day, with effector cells: target cells were 1:4 (0.25), 1:2 (0.5), 1:1 (1) or 2:1 (2) adding the genetically engineered T cells prepared in example 7 and the co-cultured untransduced T cells to wells containing target cells, respectively, under normoxic conditions (21% O 2 ) Or anoxic conditions (1%O) 2 ) Culturing for 24 hr, and using after co-culturingThe microplate luminescence detector detects luciferase activity value of target cells. The results are shown in FIG. 5.
The formula for calculating the cell killing rate is shown as follows:
cell killing rate (%) = (untransduced T cell group luciferase activity value-experimental group luciferase activity value)/untransduced T cell group luciferase activity value×100
CD47 CAR-T cells (positive control) were able to kill SKOV3 (fig. 5 a) and NCI-H292 tumor cells (fig. 5 b) effectively under normoxic or hypoxic conditions, with no apparent selective killing characteristics.
The tumor killing activity of conditionally controlled spliceable system-regulated CD47 CAR-T cells under normoxic conditions was low, and the SopE-based conditionally controlled spliceable system-regulated CD47 CAR-T cells were found to be in effector cells: target cells were 1: the killing rate in the process 1 is 3 percent (SKOV 3) and 6 percent (NCI-H292), and the SKOV3 tumor cells (84 percent) and NCI-H292 tumor cells (96 percent) can be selectively and efficiently killed in the anoxic environment, so that the killing activity of the tumor cells is improved by 28 times and 16 times. A ERm based conditionally controlled alternative splicing system regulated CD47 CAR-T cell was found in effector cells: target cells were 1: the killing rate in the process 1 is 3 percent (SKOV 3) and 6 percent (NCI-H292), so that SKOV3 tumor cells (58 percent) and NCI-H292 tumor cells (96 percent) can be selectively and efficiently killed in the anoxic environment, and the killing activity of the tumor cells is improved by 19 times and 16 times.
Example 9 in vivo anti-tumor effects of conditionally-controlled alternative splicing System-mediated CAR-T cells
The back local depilation is carried out on the B-NDG mice fed by the sterile isolator one day in advance, and the depilation can be carried out by using a depilation paste or an animal shaver to expose the skin of a tumor cell inoculation part. The left hand is used for fixing the mouse, namely, the left hand simultaneously grasps the head, neck and back skin of the mouse, the back of the mouse turns left, and after the shaving part on the right side of the back is fully exposed, the right hand is used for disinfecting the hand by using the alcohol cotton ball. After the prepared NCI-H292 tumor cells were blown and mixed by a 1mL insulin syringe, 125. Mu.L of the cell suspension (5X 10) 6 Tumor cells), the needle head is obliquely penetrated into the subcutaneous part of the mouse at an angle of 30-40 degrees with the skin, and the cell suspension is slowly injected in a pushing way, so that the cell overflow is avoided. After the injection of 125. Mu.L of the cell suspension was completed, the needle was withdrawn rapidly after 2-3 seconds, and the subcutaneous bulge at the injection site was seen as a clearly visible small bag. The tumor formation and health status of the mice were observed every 2-3 days after cell inoculation, and baseline tumor volumes were measured with vernier calipers after tumor formation and subsequent experiments were performed.
Intravenous or intratumoral injection 5X 10 at the fifth and eleventh days after tumor cell inoculation, respectively 6 The size of the tumor was measured every 2-3 days using vernier calipers for the long and short diameters of the tumor, respectively, for CAR-T cells and negative control T cells (UTD) regulated by the conditionally controlled alternative splicing system. The results are shown in FIG. 6.
The calculation formula of tumor volume is as follows: volume of= (long diameter x short diameter) 2 )/2。
Both intravenous infusion and intratumoral injection conditionally controlled spliceable system-regulated CAR-T cells were effective in controlling lung cancer growth, compared to UTD treatment group, the degrader SopE-based conditionally controlled spliceable system-regulated CAR-T cells (stable integration of EF1 alpha-BFP-CD 47-28BB-Int N -SopE-HRE-ODD-Int C- CD3z or EF1 alpha-BFP-CD 47-28BB-Int N -ERm-HRE-ODD-Int C- CD3 z) was more remarkable, and tumor inhibition rates of one month after cell reinfusion were 72.1% (intravenous reinfusion) and 85.6% (intratumoral injection), respectively.
The foregoing description of the preferred embodiment of the invention is merely illustrative of and not limiting on the invention in any way, and although the invention has been described above in terms of the preferred embodiment, it is not intended to be limiting. Any person skilled in the art may make some changes or modifications to obtain equivalent embodiments of equivalent variations without departing from the scope of the technical solution of the present invention, but any modifications, equivalent variations and modifications to the above embodiments according to the technical matter of the present invention still fall within the scope of the technical solution of the present invention.
Sequence listing
<110> Shanghai Xinwan biotechnology Co., ltd
<120> a conditionally controlled spliceable chimeric antigen receptor molecule and uses thereof
<130> DIC21110080
<140> 2021112730255
<141> 2021-10-29
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 617
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Glu Val Gln
20 25 30
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
35 40 45
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr Gly Met Ser
50 55 60
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Thr Ile
65 70 75 80
Thr Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg
85 90 95
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met
100 105 110
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser
115 120 125
Leu Ala Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
130 135 140
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
145 150 155 160
Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
165 170 175
Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser
180 185 190
Asp Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
195 200 205
Leu Ile Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe
210 215 220
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
225 230 235 240
Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn Gly His Gly Phe
245 250 255
Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ala Ser Ile
260 265 270
Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly
275 280 285
Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe
290 295 300
Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val
305 310 315 320
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp
325 330 335
Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
340 345 350
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
355 360 365
Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Phe Ser Val Val Lys
370 375 380
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
385 390 395 400
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
405 410 415
Glu Glu Glu Glu Gly Gly Cys Glu Leu Thr Arg Ser Gly Tyr Cys Leu
420 425 430
Asp Leu Lys Thr Gln Val Gln Thr Pro Gln Gly Met Lys Glu Ile Ser
435 440 445
Asn Ile Gln Val Gly Asp Leu Val Leu Ser Asn Thr Gly Tyr Asn Glu
450 455 460
Val Leu Asn Val Phe Pro Lys Ser Lys Lys Lys Ser Tyr Lys Ile Thr
465 470 475 480
Leu Glu Asp Gly Lys Glu Ile Ile Cys Ser Glu Glu His Leu Phe Pro
485 490 495
Thr Gln Thr Gly Glu Met Asn Ile Ser Gly Gly Leu Lys Glu Gly Met
500 505 510
Cys Leu Tyr Val Lys Glu Thr Lys Ile Thr Leu Ser Pro Gln Asn Phe
515 520 525
Arg Ile Gln Lys Gln Glu Thr Thr Leu Leu Lys Glu Lys Ser Thr Glu
530 535 540
Lys Asn Ser Leu Ala Lys Ser Ile Leu Ala Val Lys Asn His Phe Ile
545 550 555 560
Glu Leu Arg Ser Lys Leu Ser Glu Arg Phe Ile Ser His Lys Asn Thr
565 570 575
Glu Ser Ser Ala Thr His Phe His Arg Gly Ser Ala Ser Glu Gly Arg
580 585 590
Ala Val Leu Thr Asn Lys Val Val Lys Asp Phe Met Leu Gln Thr Leu
595 600 605
Asn Asp Ile Asp Ile Arg Gly Ser Ala
610 615
<210> 2
<211> 763
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Glu Val Gln
20 25 30
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
35 40 45
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr Gly Met Ser
50 55 60
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Thr Ile
65 70 75 80
Thr Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg
85 90 95
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met
100 105 110
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser
115 120 125
Leu Ala Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
130 135 140
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
145 150 155 160
Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
165 170 175
Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser
180 185 190
Asp Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
195 200 205
Leu Ile Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe
210 215 220
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
225 230 235 240
Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn Gly His Gly Phe
245 250 255
Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ala Ser Ile
260 265 270
Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly
275 280 285
Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe
290 295 300
Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val
305 310 315 320
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp
325 330 335
Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
340 345 350
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
355 360 365
Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Phe Ser Val Val Lys
370 375 380
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
385 390 395 400
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
405 410 415
Glu Glu Glu Glu Gly Gly Cys Glu Leu Thr Arg Ser Gly Tyr Cys Leu
420 425 430
Asp Leu Lys Thr Gln Val Gln Thr Pro Gln Gly Met Lys Glu Ile Ser
435 440 445
Asn Ile Gln Val Gly Asp Leu Val Leu Ser Asn Thr Gly Tyr Asn Glu
450 455 460
Val Leu Asn Val Phe Pro Lys Ser Lys Lys Lys Ser Tyr Lys Ile Thr
465 470 475 480
Leu Glu Asp Gly Lys Glu Ile Ile Cys Ser Glu Glu His Leu Phe Pro
485 490 495
Thr Gln Thr Gly Glu Met Asn Ile Ser Gly Gly Leu Lys Glu Gly Met
500 505 510
Cys Leu Tyr Val Lys Glu Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln
515 520 525
Met Val Ser Ala Leu Leu Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu
530 535 540
Tyr Asp Pro Thr Arg Pro Phe Ser Glu Ala Ser Met Met Gly Leu Leu
545 550 555 560
Thr Asn Leu Ala Asp Arg Glu Leu Val His Met Ile Asn Trp Ala Lys
565 570 575
Arg Val Pro Gly Phe Val Asp Leu Ala Leu His Asp Gln Val His Leu
580 585 590
Leu Glu Cys Ala Trp Met Glu Ile Leu Met Ile Gly Leu Val Trp Arg
595 600 605
Ser Met Glu His Pro Gly Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu
610 615 620
Asp Arg Asn Gln Gly Lys Cys Val Glu Gly Gly Val Glu Ile Phe Asp
625 630 635 640
Met Leu Leu Ala Thr Ser Ser Arg Phe Arg Met Met Asn Leu Gln Gly
645 650 655
Glu Glu Phe Val Cys Leu Lys Ser Ile Ile Leu Leu Asn Ser Gly Val
660 665 670
Tyr Thr Phe Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu Lys Asp His
675 680 685
Ile His Arg Val Leu Asp Lys Ile Thr Asp Thr Leu Ile His Leu Met
690 695 700
Ala Lys Ala Gly Leu Thr Leu Gln Gln Gln His Gln Arg Leu Ala Gln
705 710 715 720
Leu Leu Leu Ile Leu Ser His Ile Arg His Met Ser Ser Lys Arg Met
725 730 735
Glu His Leu Tyr Ser Met Lys Cys Lys Asn Val Val Pro Leu Ser Asp
740 745 750
Leu Leu Leu Glu Met Leu Asp Ala His Arg Leu
755 760
<210> 3
<211> 379
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Met Ser Glu Asp Thr Ser Ser Leu Phe Asp Lys Leu Lys Lys Glu Pro
1 5 10 15
Asp Ala Leu Thr Leu Leu Ala Pro Ala Ala Gly Asp Thr Ile Ile Ser
20 25 30
Leu Asp Phe Gly Ser Asn Asp Thr Glu Thr Asp Asp Gln Gln Leu Glu
35 40 45
Glu Val Pro Leu Tyr Asn Asp Val Met Leu Pro Ser Pro Asn Glu Lys
50 55 60
Leu Gln Asn Ile Asn Leu Ala Met Ser Pro Leu Pro Thr Ala Glu Thr
65 70 75 80
Pro Lys Pro Leu Arg Ser Ser Ala Asp Pro Ala Leu Asn Gln Glu Val
85 90 95
Ala Leu Lys Leu Glu Pro Asn Pro Glu Ser Leu Glu Leu Ser Phe Thr
100 105 110
Met Pro Gln Ile Gln Asp Gln Thr Pro Ser Pro Ser Asp Gly Ser Thr
115 120 125
Arg Gln Ser Ser Pro Glu Pro Asn Ser Pro Ser Glu Tyr Cys Phe Tyr
130 135 140
Val Asp Ser Asp Met Val Asn Glu Phe Lys Leu Glu Leu Val Glu Lys
145 150 155 160
Leu Phe Ala Glu Asp Thr Glu Ala Lys Asn Pro Phe Ser Thr Gln Asp
165 170 175
Thr Asp Leu Asp Leu Glu Met Leu Ala Pro Tyr Ile Pro Met Asp Asp
180 185 190
Asp Phe Gln Leu Arg Ser Phe Asp Gln Leu Ser Pro Leu Glu Ser Ser
195 200 205
Ser Ala Ser Pro Glu Ser Ala Ser Pro Gln Ser Thr Val Thr Val Phe
210 215 220
Gln Leu Lys Lys Ile Leu Lys Ile Glu Glu Leu Asp Glu Arg Glu Leu
225 230 235 240
Ile Asp Ile Glu Val Ser Gly Asn His Leu Phe Tyr Ala Asn Asp Ile
245 250 255
Leu Thr His Asn Ser Ser Ser Ser Asp Val Arg Val Lys Phe Ser Arg
260 265 270
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
275 280 285
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
290 295 300
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn
305 310 315 320
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
325 330 335
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
340 345 350
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
355 360 365
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
370 375
<210> 4
<211> 1453
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
ccacagtgca tacgtgggct ccaacaggtc ctcttccaca gtgcatacgt gggctccaac 60
aggtcctctt ccacagtgca tacgtgggct ccaacaggtc ctcttccaca gtgcatacgt 120
gggctccaac aggtcctctt ccacagtgca tacgtgggct ccaacaggtc ctcttaccaa 180
aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt 240
aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc 300
ttctagagcc accatgagcg aggacaccag cagcctgttc gacaagctga agaaggagcc 360
cgacgccctg accctgctgg cccctgctgc tggagacacc atcatctccc tggacttcgg 420
cagcaacgac accgagaccg acgaccagca gctggaggag gtgcccctgt acaacgacgt 480
gatgctgccc tctcccaacg aaaaactgca gaacatcaac ctggctatga gccccctgcc 540
caccgccgaa acaccaaaac ccctgagatc cagcgccgac cccgccctga accaggaagt 600
ggccctgaaa ctggaaccca accccgagag cctggagctg agcttcacca tgccccagat 660
ccaggaccag acccccagcc ccagcgacgg aagcaccaga cagagcagcc ccgagcctaa 720
ctcccccagc gaatactgct tctatgtgga cagcgacatg gtgaacgagt tcaagctgga 780
gctggtggaa aaactgttcg ccgaggacac agaagccaaa aaccccttca gcacccagga 840
cacagacctg gacctggaga tgctggcccc ctacatcccc atggacgacg acttccagct 900
gagatccttc gaccagctga gccccctgga aagcagcagc gcctcccccg aatcagccag 960
cccccagagc accgtgaccg tgttccagct gaaaaagatc ctgaagatcg aggagctgga 1020
cgagcgggaa ctgatcgaca tcgaagtgtc cggaaaccac ctgttctacg ccaacgacat 1080
cctgacacac aatagcagca gcagcgacgt gagagtgaaa ttcagcagaa gcgccgacgc 1140
ccccgcctac cagcagggac agaatcagct gtacaacgaa ctgaacctgg gcagaaggga 1200
ggaatacgac gtgctggaca agaggagagg aagggacccc gagatgggag gaaaaccaca 1260
gagaagaaag aacccacagg agggactgta caacgagctg cagaaggaca agatggccga 1320
ggcctacagt gaaattggca tgaagggaga gagaagaaga ggaaagggac acgacggcct 1380
gtaccagggc ctgagcaccg ctaccaagga cacatacgac gccctgcaca tgcaggccct 1440
gccaccaaga tga 1453
<210> 5
<211> 4673
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60
gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120
atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180
tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 240
tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 300
cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg 360
tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc 420
tggcttgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctccct 480
gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc 540
tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg 600
gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg 660
cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg 720
cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca 780
ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg 840
aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt 900
ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc 960
gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg 1020
atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg 1080
taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag 1140
acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggatctat ttccggtgag 1200
acccaagctg gctagcgcca ccatggctct gccagtgaca gctctcctcc tcccactcgc 1260
cctgctgctg cacgccgcta gacctgacta caaggacgac gacgacaagg aggtgcagct 1320
ggtggagtct gggggaggct tggtccagcc tggggggtcc ctgagactct cctgtgcagc 1380
ctctggattc acctttagtg gctatggcat gagctgggtc cgccaggctc cagggaaggg 1440
gctggagtgg gtggccacca taactagtgg tggaacttac acctactatc cagactctgt 1500
gaagggccga ttcaccatct ccagagacaa cgccaagaac tcactgtatc tgcaaatgaa 1560
cagcctgaga gccgaggaca cggctgtgta ttactgtgcg agatccctcg cgggaaatgc 1620
tatggactac tggggccaag gaaccctggt caccgtctcc tcaggcggag gcggcagtgg 1680
cgggggcggg tccggcggag gcgggagcga aattgtgttg acacagtctc cagccaccct 1740
gtctttgtct ccaggggaaa gagccaccct ctcctgcagg gccagtcaga ctattagcga 1800
ctacttacac tggtaccaac agaaacctgg ccaggctccc aggctcctca tcaaatttgc 1860
atcccaatcc atttctggca tcccagccag gttcagtggc agtgggtctg ggacagactt 1920
cactctcacc atcagcagcc tagagcctga agattttgca gtttattact gtcagaatgg 1980
tcacggcttt cctcggacgt tcggccaagg gaccaaggtg gaaatcaaag ctagcatcga 2040
ggtgatgtac cctccccctt acctggacaa cgagaagagc aacggcacca tcatccacgt 2100
gaagggcaag cacctgtgcc ctagccccct gttccccgga cctagcaagc ccttttgggt 2160
gctggtggtg gtgggcggcg tgctggcctg ttactccctg ctggtgaccg tggccttcat 2220
tatcttctgg gtgaggagca agaggagcag gctgctgcac agcgactaca tgaacatgac 2280
acccaggaga cctggcccca ccagaaagca ctaccagccc tatgcccccc ccagagactt 2340
tgccgcctac agaagcaggt tcagcgtggt gaagaggggc aggaagaagc tgctgtacat 2400
cttcaagcag cccttcatga ggcccgtgca gaccacccag gaggaggacg gctgcagctg 2460
caggttcccc gaggaggagg aaggcggatg cgagctgacc cggtctggct actgcctcga 2520
cctcaagacc caggtgcaga cccctcaggg catgaaggag atttctaaca ttcaggtggg 2580
cgacctcgtg ctgagcaaca ccggctacaa cgaggtgctc aacgtgttcc caaagtctaa 2640
gaagaagtct tacaagatca cactggagga cggcaaggag attatttgct ctgaggagca 2700
cctgttccct acccagacag gcgagatgaa catttctggc ggcctcaagg agggcatgtg 2760
cctgtacgtg aaggagacca agatcaccct gagcccccag aacttccgca tccagaagca 2820
ggagaccacc ctgctgaagg agaagagcac cgagaagaac agcctggcca agagcatcct 2880
ggccgtgaag aaccacttca tcgagctgcg cagcaagctg agcgagcgct tcatcagcca 2940
caagaacacc gagagcagcg ccacccactt ccaccgcggc agcgccagcg agggccgcgc 3000
cgtgctgacc aacaaggtgg tgaaggactt catgctgcag accctgaacg acatcgacat 3060
ccgcggcagc gcctgaacgc gtaaatgtgg tatggctgat tatgatccga tatctccagt 3120
gtggtgtgca ggatcccagc acagtctcca ggcgatctga cggttcacta aacgagctct 3180
gcttatatag gcctcccacc gtacacgcca cctcgacata ccacagtgca tacgtgggct 3240
ccaacaggtc ctcttccaca gtgcatacgt gggctccaac aggtcctctt ccacagtgca 3300
tacgtgggct ccaacaggtc ctcttccaca gtgcatacgt gggctccaac aggtcctctt 3360
ccacagtgca tacgtgggct ccaacaggtc ctcttaccaa aatcaacggg actttccaaa 3420
atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt 3480
ctatataagc agagctggtt tagtgaaccg tcagatccgc ttctagagcc accatgagcg 3540
aggacaccag cagcctgttc gacaagctga agaaggagcc cgacgccctg accctgctgg 3600
cccctgctgc tggagacacc atcatctccc tggacttcgg cagcaacgac accgagaccg 3660
acgaccagca gctggaggag gtgcccctgt acaacgacgt gatgctgccc tctcccaacg 3720
aaaaactgca gaacatcaac ctggctatga gccccctgcc caccgccgaa acaccaaaac 3780
ccctgagatc cagcgccgac cccgccctga accaggaagt ggccctgaaa ctggaaccca 3840
accccgagag cctggagctg agcttcacca tgccccagat ccaggaccag acccccagcc 3900
ccagcgacgg aagcaccaga cagagcagcc ccgagcctaa ctcccccagc gaatactgct 3960
tctatgtgga cagcgacatg gtgaacgagt tcaagctgga gctggtggaa aaactgttcg 4020
ccgaggacac agaagccaaa aaccccttca gcacccagga cacagacctg gacctggaga 4080
tgctggcccc ctacatcccc atggacgacg acttccagct gagatccttc gaccagctga 4140
gccccctgga aagcagcagc gcctcccccg aatcagccag cccccagagc accgtgaccg 4200
tgttccagct gaaaaagatc ctgaagatcg aggagctgga cgagcgggaa ctgatcgaca 4260
tcgaagtgtc cggaaaccac ctgttctacg ccaacgacat cctgacacac aatagcagca 4320
gcagcgacgt gagagtgaaa ttcagcagaa gcgccgacgc ccccgcctac cagcagggac 4380
agaatcagct gtacaacgaa ctgaacctgg gcagaaggga ggaatacgac gtgctggaca 4440
agaggagagg aagggacccc gagatgggag gaaaaccaca gagaagaaag aacccacagg 4500
agggactgta caacgagctg cagaaggaca agatggccga ggcctacagt gaaattggca 4560
tgaagggaga gagaagaaga ggaaagggac acgacggcct gtaccagggc ctgagcaccg 4620
ctaccaagga cacatacgac gccctgcaca tgcaggccct gccaccaaga tga 4673
<210> 6
<211> 5111
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60
gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120
atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180
tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 240
tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 300
cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg 360
tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc 420
tggcttgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctccct 480
gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc 540
tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg 600
gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg 660
cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg 720
cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca 780
ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg 840
aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt 900
ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc 960
gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg 1020
atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg 1080
taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag 1140
acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggatctat ttccggtgag 1200
acccaagctg gctagcgcca ccatggctct gccagtgaca gctctcctcc tcccactcgc 1260
cctgctgctg cacgccgcta gacctgacta caaggacgac gacgacaagg aggtgcagct 1320
ggtggagtct gggggaggct tggtccagcc tggggggtcc ctgagactct cctgtgcagc 1380
ctctggattc acctttagtg gctatggcat gagctgggtc cgccaggctc cagggaaggg 1440
gctggagtgg gtggccacca taactagtgg tggaacttac acctactatc cagactctgt 1500
gaagggccga ttcaccatct ccagagacaa cgccaagaac tcactgtatc tgcaaatgaa 1560
cagcctgaga gccgaggaca cggctgtgta ttactgtgcg agatccctcg cgggaaatgc 1620
tatggactac tggggccaag gaaccctggt caccgtctcc tcaggcggag gcggcagtgg 1680
cgggggcggg tccggcggag gcgggagcga aattgtgttg acacagtctc cagccaccct 1740
gtctttgtct ccaggggaaa gagccaccct ctcctgcagg gccagtcaga ctattagcga 1800
ctacttacac tggtaccaac agaaacctgg ccaggctccc aggctcctca tcaaatttgc 1860
atcccaatcc atttctggca tcccagccag gttcagtggc agtgggtctg ggacagactt 1920
cactctcacc atcagcagcc tagagcctga agattttgca gtttattact gtcagaatgg 1980
tcacggcttt cctcggacgt tcggccaagg gaccaaggtg gaaatcaaag ctagcatcga 2040
ggtgatgtac cctccccctt acctggacaa cgagaagagc aacggcacca tcatccacgt 2100
gaagggcaag cacctgtgcc ctagccccct gttccccgga cctagcaagc ccttttgggt 2160
gctggtggtg gtgggcggcg tgctggcctg ttactccctg ctggtgaccg tggccttcat 2220
tatcttctgg gtgaggagca agaggagcag gctgctgcac agcgactaca tgaacatgac 2280
acccaggaga cctggcccca ccagaaagca ctaccagccc tatgcccccc ccagagactt 2340
tgccgcctac agaagcaggt tcagcgtggt gaagaggggc aggaagaagc tgctgtacat 2400
cttcaagcag cccttcatga ggcccgtgca gaccacccag gaggaggacg gctgcagctg 2460
caggttcccc gaggaggagg aaggcggatg cgagctgacc cggtctggct actgcctcga 2520
cctcaagacc caggtgcaga cccctcaggg catgaaggag atttctaaca ttcaggtggg 2580
cgacctcgtg ctgagcaaca ccggctacaa cgaggtgctc aacgtgttcc caaagtctaa 2640
gaagaagtct tacaagatca cactggagga cggcaaggag attatttgct ctgaggagca 2700
cctgttccct acccagacag gcgagatgaa catttctggc ggcctcaagg agggcatgtg 2760
cctgtacgtg aaggagtctc tggccctgtc cctcacagcc gaccagatgg tgtccgccct 2820
cctggacgcc gagccaccaa ttctgtactc tgagtacgac ccaacacgcc ctttcagcga 2880
ggcctctatg atgggcctcc tcacaaacct cgccgaccgg gagctggtgc acatgattaa 2940
ctgggccaag agagtgcccg gcttcgtgga cctcgccctg cacgaccagg tgcacctgct 3000
ggagtgcgcc tggatggaga tcctcatgat tggcctggtg tggcggtcta tggagcaccc 3060
aggcaagctg ctgttcgccc ctaacctcct gctcgaccgc aaccagggca agtgcgtgga 3120
gggcggcgtg gagattttcg acatgctcct cgccacatct agccggttcc ggatgatgaa 3180
cctccaaggc gaggagttcg tgtgcctgaa gtctattatt ctgctcaact ctggcgtgta 3240
caccttcctg tcttctacac tcaagtctct ggaggagaag gaccacattc accgcgtgct 3300
cgacaagatt accgacacac tcattcacct gatggccaag gccggcctca cactgcaaca 3360
gcagcaccag agactggccc agctgctcct gatcctgtcc cacattaggc acatgtcttc 3420
taagcgcatg gagcacctgt actctatgaa gtgcaagaac gtggtgccac tgtctgacct 3480
gctcctggaa atgctggacg cccaccggct gtgaacgcgt aaatgtggta tggctgatta 3540
tgatccgata tctccagtgt ggtgtgcagg atcccagcac agtctccagg cgatctgacg 3600
gttcactaaa cgagctctgc ttatataggc ctcccaccgt acacgccacc tcgacatacc 3660
acagtgcata cgtgggctcc aacaggtcct cttccacagt gcatacgtgg gctccaacag 3720
gtcctcttcc acagtgcata cgtgggctcc aacaggtcct cttccacagt gcatacgtgg 3780
gctccaacag gtcctcttcc acagtgcata cgtgggctcc aacaggtcct cttaccaaaa 3840
tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag 3900
gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgctt 3960
ctagagccac catgagcgag gacaccagca gcctgttcga caagctgaag aaggagcccg 4020
acgccctgac cctgctggcc cctgctgctg gagacaccat catctccctg gacttcggca 4080
gcaacgacac cgagaccgac gaccagcagc tggaggaggt gcccctgtac aacgacgtga 4140
tgctgccctc tcccaacgaa aaactgcaga acatcaacct ggctatgagc cccctgccca 4200
ccgccgaaac accaaaaccc ctgagatcca gcgccgaccc cgccctgaac caggaagtgg 4260
ccctgaaact ggaacccaac cccgagagcc tggagctgag cttcaccatg ccccagatcc 4320
aggaccagac ccccagcccc agcgacggaa gcaccagaca gagcagcccc gagcctaact 4380
cccccagcga atactgcttc tatgtggaca gcgacatggt gaacgagttc aagctggagc 4440
tggtggaaaa actgttcgcc gaggacacag aagccaaaaa ccccttcagc acccaggaca 4500
cagacctgga cctggagatg ctggccccct acatccccat ggacgacgac ttccagctga 4560
gatccttcga ccagctgagc cccctggaaa gcagcagcgc ctcccccgaa tcagccagcc 4620
cccagagcac cgtgaccgtg ttccagctga aaaagatcct gaagatcgag gagctggacg 4680
agcgggaact gatcgacatc gaagtgtccg gaaaccacct gttctacgcc aacgacatcc 4740
tgacacacaa tagcagcagc agcgacgtga gagtgaaatt cagcagaagc gccgacgccc 4800
ccgcctacca gcagggacag aatcagctgt acaacgaact gaacctgggc agaagggagg 4860
aatacgacgt gctggacaag aggagaggaa gggaccccga gatgggagga aaaccacaga 4920
gaagaaagaa cccacaggag ggactgtaca acgagctgca gaaggacaag atggccgagg 4980
cctacagtga aattggcatg aagggagaga gaagaagagg aaagggacac gacggcctgt 5040
accagggcct gagcaccgct accaaggaca catacgacgc cctgcacatg caggccctgc 5100
caccaagatg a 5111
<210> 7
<211> 1614
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atggctctgc cagtgacagc tctcctcctc ccactcgccc tgctgctgca cgccgctaga 60
cctgactaca aggacgacga cgacaaggag gtgcagctgg tggagtctgg gggaggcttg 120
gtccagcctg gggggtccct gagactctcc tgtgcagcct ctggattcac ctttagtggc 180
tatggcatga gctgggtccg ccaggctcca gggaaggggc tggagtgggt ggccaccata 240
actagtggtg gaacttacac ctactatcca gactctgtga agggccgatt caccatctcc 300
agagacaacg ccaagaactc actgtatctg caaatgaaca gcctgagagc cgaggacacg 360
gctgtgtatt actgtgcgag atccctcgcg ggaaatgcta tggactactg gggccaagga 420
accctggtca ccgtctcctc aggcggaggc ggcagtggcg ggggcgggtc cggcggaggc 480
gggagcgaaa ttgtgttgac acagtctcca gccaccctgt ctttgtctcc aggggaaaga 540
gccaccctct cctgcagggc cagtcagact attagcgact acttacactg gtaccaacag 600
aaacctggcc aggctcccag gctcctcatc aaatttgcat cccaatccat ttctggcatc 660
ccagccaggt tcagtggcag tgggtctggg acagacttca ctctcaccat cagcagccta 720
gagcctgaag attttgcagt ttattactgt cagaatggtc acggctttcc tcggacgttc 780
ggccaaggga ccaaggtgga aatcaaagct agcatcgagg tgatgtaccc tcccccttac 840
ctggacaacg agaagagcaa cggcaccatc atccacgtga agggcaagca cctgtgccct 900
agccccctgt tccccggacc tagcaagccc ttttgggtgc tggtggtggt gggcggcgtg 960
ctggcctgtt actccctgct ggtgaccgtg gccttcatta tcttctgggt gaggagcaag 1020
aggagcaggc tgctgcacag cgactacatg aacatgacac ccaggagacc tggccccacc 1080
agaaagcact accagcccta tgcccccccc agagactttg ccgcctacag aagcaggttc 1140
agcgtggtga agaggggcag gaagaagctg ctgtacatct tcaagcagcc cttcatgagg 1200
cccgtgcaga ccacccagga ggaggacggc tgcagctgca ggttccccga ggaggaggaa 1260
ggcggatgcg agctgagagt gaagttctcc agaagcgctg acgcccctgc ctaccagcag 1320
ggacagaacc agctgtataa cgagctgaac ctgggcagga gagaggagta cgatgtcctg 1380
gacaagagga gaggacgtga tcctgagatg ggcggcaagc cccaaaggag aaagaacccc 1440
caggagggac tgtacaatga gctgcagaag gacaagatgg ccgaggccta ctccgaaatc 1500
ggcatgaaag gcgagaggag aaggggcaaa ggccacgatg gcctgtacca gggcctgagc 1560
acagccacca aagacacata cgacgccctg cacatgcagg ccctgccccc tagg 1614
<210> 8
<211> 2337
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
atggctctgc cagtgacagc tctcctcctc ccactcgccc tgctgctgca cgccgctaga 60
cctgactaca aggacgacga cgacaaggag gtgcagctgg tggagtctgg gggaggcttg 120
gtccagcctg gggggtccct gagactctcc tgtgcagcct ctggattcac ctttagtggc 180
tatggcatga gctgggtccg ccaggctcca gggaaggggc tggagtgggt ggccaccata 240
actagtggtg gaacttacac ctactatcca gactctgtga agggccgatt caccatctcc 300
agagacaacg ccaagaactc actgtatctg caaatgaaca gcctgagagc cgaggacacg 360
gctgtgtatt actgtgcgag atccctcgcg ggaaatgcta tggactactg gggccaagga 420
accctggtca ccgtctcctc aggcggaggc ggcagtggcg ggggcgggtc cggcggaggc 480
gggagcgaaa ttgtgttgac acagtctcca gccaccctgt ctttgtctcc aggggaaaga 540
gccaccctct cctgcagggc cagtcagact attagcgact acttacactg gtaccaacag 600
aaacctggcc aggctcccag gctcctcatc aaatttgcat cccaatccat ttctggcatc 660
ccagccaggt tcagtggcag tgggtctggg acagacttca ctctcaccat cagcagccta 720
gagcctgaag attttgcagt ttattactgt cagaatggtc acggctttcc tcggacgttc 780
ggccaaggga ccaaggtgga aatcaaagct agcatcgagg tgatgtaccc tcccccttac 840
ctggacaacg agaagagcaa cggcaccatc atccacgtga agggcaagca cctgtgccct 900
agccccctgt tccccggacc tagcaagccc ttttgggtgc tggtggtggt gggcggcgtg 960
ctggcctgtt actccctgct ggtgaccgtg gccttcatta tcttctgggt gaggagcaag 1020
aggagcaggc tgctgcacag cgactacatg aacatgacac ccaggagacc tggccccacc 1080
agaaagcact accagcccta tgcccccccc agagactttg ccgcctacag aagcaggttc 1140
agcgtggtga agaggggcag gaagaagctg ctgtacatct tcaagcagcc cttcatgagg 1200
cccgtgcaga ccacccagga ggaggacggc tgcagctgca ggttccccga ggaggaggaa 1260
ggcggatgcg agctgagagt gaagttctcc agaagcgctg acgcccctgc ctaccagcag 1320
ggacagaacc agctgtataa cgagctgaac ctgggcagga gagaggagta cgatgtcctg 1380
gacaagagga gaggacgtga tcctgagatg ggcggcaagc cccaaaggag aaagaacccc 1440
caggagggac tgtacaatga gctgcagaag gacaagatgg ccgaggccta ctccgaaatc 1500
ggcatgaaag gcgagaggag aaggggcaaa ggccacgatg gcctgtacca gggcctgagc 1560
acagccacca aagacacata cgacgccctg cacatgcagg ccctgccccc taggctcgag 1620
ggcggaggcg ggtccggggg cgggggctcc ggcggcggag ggtccagcga ggatacctca 1680
tcactgtttg acaagctcaa gaaggagccc gacgcactca cactcctcgc acccgccgcc 1740
ggcgatacaa ttattagcct ggactttggg tctaacgata cagagaccga cgaccagcag 1800
ctggaggagg tgcctctgta caatgatgtc atgctgcctt ctcctaatga gaagctgcaa 1860
aatattaatc tggctatgtc accactgcct accgccgaga cacctaagcc actcaggtct 1920
agcgccgatc ccgctctgaa ccaggaggtg gccctgaagc tagagccaaa cccagagtct 1980
ctggagcttt catttactat gccacagatt caggatcaga ctccatctcc atccgacgga 2040
tcaactaggc agtcttcccc tgagcccaat tccccatccg agtactgctt ctacgtcgat 2100
agcgacatgg tgaatgagtt taagctggag ttagtcgaga agctgttcgc agaggatact 2160
gaggcaaaga acccattttc aacacaggac accgacctgg acttagaaat gctcgcacct 2220
tacattccta tggacgatga ctttcagctg cggtcatttg atcagctgtc ccctctcgaa 2280
tcttctagcg caagtcctga gagcgcttca ccacagtcaa ccgtgaccgt gttccag 2337
<210> 9
<211> 1854
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
atggctctgc cagtgacagc tctcctcctc ccactcgccc tgctgctgca cgccgctaga 60
cctgactaca aggacgacga cgacaaggag gtgcagctgg tggagtctgg gggaggcttg 120
gtccagcctg gggggtccct gagactctcc tgtgcagcct ctggattcac ctttagtggc 180
tatggcatga gctgggtccg ccaggctcca gggaaggggc tggagtgggt ggccaccata 240
actagtggtg gaacttacac ctactatcca gactctgtga agggccgatt caccatctcc 300
agagacaacg ccaagaactc actgtatctg caaatgaaca gcctgagagc cgaggacacg 360
gctgtgtatt actgtgcgag atccctcgcg ggaaatgcta tggactactg gggccaagga 420
accctggtca ccgtctcctc aggcggaggc ggcagtggcg ggggcgggtc cggcggaggc 480
gggagcgaaa ttgtgttgac acagtctcca gccaccctgt ctttgtctcc aggggaaaga 540
gccaccctct cctgcagggc cagtcagact attagcgact acttacactg gtaccaacag 600
aaacctggcc aggctcccag gctcctcatc aaatttgcat cccaatccat ttctggcatc 660
ccagccaggt tcagtggcag tgggtctggg acagacttca ctctcaccat cagcagccta 720
gagcctgaag attttgcagt ttattactgt cagaatggtc acggctttcc tcggacgttc 780
ggccaaggga ccaaggtgga aatcaaagct agcatcgagg tgatgtaccc tcccccttac 840
ctggacaacg agaagagcaa cggcaccatc atccacgtga agggcaagca cctgtgccct 900
agccccctgt tccccggacc tagcaagccc ttttgggtgc tggtggtggt gggcggcgtg 960
ctggcctgtt actccctgct ggtgaccgtg gccttcatta tcttctgggt gaggagcaag 1020
aggagcaggc tgctgcacag cgactacatg aacatgacac ccaggagacc tggccccacc 1080
agaaagcact accagcccta tgcccccccc agagactttg ccgcctacag aagcaggttc 1140
agcgtggtga agaggggcag gaagaagctg ctgtacatct tcaagcagcc cttcatgagg 1200
cccgtgcaga ccacccagga ggaggacggc tgcagctgca ggttccccga ggaggaggaa 1260
ggcggatgcg agctgacccg gtctggctac tgcctcgacc tcaagaccca ggtgcagacc 1320
cctcagggca tgaaggagat ttctaacatt caggtgggcg acctcgtgct gagcaacacc 1380
ggctacaacg aggtgctcaa cgtgttccca aagtctaaga agaagtctta caagatcaca 1440
ctggaggacg gcaaggagat tatttgctct gaggagcacc tgttccctac ccagacaggc 1500
gagatgaaca tttctggcgg cctcaaggag ggcatgtgcc tgtacgtgaa ggagaccaag 1560
atcaccctga gcccccagaa cttccgcatc cagaagcagg agaccaccct gctgaaggag 1620
aagagcaccg agaagaacag cctggccaag agcatcctgg ccgtgaagaa ccacttcatc 1680
gagctgcgca gcaagctgag cgagcgcttc atcagccaca agaacaccga gagcagcgcc 1740
acccacttcc accgcggcag cgccagcgag ggccgcgccg tgctgaccaa caaggtggtg 1800
aaggacttca tgctgcagac cctgaacgac atcgacatcc gcggcagcgc ctga 1854
<210> 10
<211> 2292
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
atggctctgc cagtgacagc tctcctcctc ccactcgccc tgctgctgca cgccgctaga 60
cctgactaca aggacgacga cgacaaggag gtgcagctgg tggagtctgg gggaggcttg 120
gtccagcctg gggggtccct gagactctcc tgtgcagcct ctggattcac ctttagtggc 180
tatggcatga gctgggtccg ccaggctcca gggaaggggc tggagtgggt ggccaccata 240
actagtggtg gaacttacac ctactatcca gactctgtga agggccgatt caccatctcc 300
agagacaacg ccaagaactc actgtatctg caaatgaaca gcctgagagc cgaggacacg 360
gctgtgtatt actgtgcgag atccctcgcg ggaaatgcta tggactactg gggccaagga 420
accctggtca ccgtctcctc aggcggaggc ggcagtggcg ggggcgggtc cggcggaggc 480
gggagcgaaa ttgtgttgac acagtctcca gccaccctgt ctttgtctcc aggggaaaga 540
gccaccctct cctgcagggc cagtcagact attagcgact acttacactg gtaccaacag 600
aaacctggcc aggctcccag gctcctcatc aaatttgcat cccaatccat ttctggcatc 660
ccagccaggt tcagtggcag tgggtctggg acagacttca ctctcaccat cagcagccta 720
gagcctgaag attttgcagt ttattactgt cagaatggtc acggctttcc tcggacgttc 780
ggccaaggga ccaaggtgga aatcaaagct agcatcgagg tgatgtaccc tcccccttac 840
ctggacaacg agaagagcaa cggcaccatc atccacgtga agggcaagca cctgtgccct 900
agccccctgt tccccggacc tagcaagccc ttttgggtgc tggtggtggt gggcggcgtg 960
ctggcctgtt actccctgct ggtgaccgtg gccttcatta tcttctgggt gaggagcaag 1020
aggagcaggc tgctgcacag cgactacatg aacatgacac ccaggagacc tggccccacc 1080
agaaagcact accagcccta tgcccccccc agagactttg ccgcctacag aagcaggttc 1140
agcgtggtga agaggggcag gaagaagctg ctgtacatct tcaagcagcc cttcatgagg 1200
cccgtgcaga ccacccagga ggaggacggc tgcagctgca ggttccccga ggaggaggaa 1260
ggcggatgcg agctgacccg gtctggctac tgcctcgacc tcaagaccca ggtgcagacc 1320
cctcagggca tgaaggagat ttctaacatt caggtgggcg acctcgtgct gagcaacacc 1380
ggctacaacg aggtgctcaa cgtgttccca aagtctaaga agaagtctta caagatcaca 1440
ctggaggacg gcaaggagat tatttgctct gaggagcacc tgttccctac ccagacaggc 1500
gagatgaaca tttctggcgg cctcaaggag ggcatgtgcc tgtacgtgaa ggagtctctg 1560
gccctgtccc tcacagccga ccagatggtg tccgccctcc tggacgccga gccaccaatt 1620
ctgtactctg agtacgaccc aacacgccct ttcagcgagg cctctatgat gggcctcctc 1680
acaaacctcg ccgaccggga gctggtgcac atgattaact gggccaagag agtgcccggc 1740
ttcgtggacc tcgccctgca cgaccaggtg cacctgctgg agtgcgcctg gatggagatc 1800
ctcatgattg gcctggtgtg gcggtctatg gagcacccag gcaagctgct gttcgcccct 1860
aacctcctgc tcgaccgcaa ccagggcaag tgcgtggagg gcggcgtgga gattttcgac 1920
atgctcctcg ccacatctag ccggttccgg atgatgaacc tccaaggcga ggagttcgtg 1980
tgcctgaagt ctattattct gctcaactct ggcgtgtaca ccttcctgtc ttctacactc 2040
aagtctctgg aggagaagga ccacattcac cgcgtgctcg acaagattac cgacacactc 2100
attcacctga tggccaaggc cggcctcaca ctgcaacagc agcaccagag actggcccag 2160
ctgctcctga tcctgtccca cattaggcac atgtcttcta agcgcatgga gcacctgtac 2220
tctatgaagt gcaagaacgt ggtgccactg tctgacctgc tcctggaaat gctggacgcc 2280
caccggctgt ga 2292
<210> 11
<211> 1140
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
atgagcgagg acaccagcag cctgttcgac aagctgaaga aggagcccga cgccctgacc 60
ctgctggccc ctgctgctgg agacaccatc atctccctgg acttcggcag caacgacacc 120
gagaccgacg accagcagct ggaggaggtg cccctgtaca acgacgtgat gctgccctct 180
cccaacgaaa aactgcagaa catcaacctg gctatgagcc ccctgcccac cgccgaaaca 240
ccaaaacccc tgagatccag cgccgacccc gccctgaacc aggaagtggc cctgaaactg 300
gaacccaacc ccgagagcct ggagctgagc ttcaccatgc cccagatcca ggaccagacc 360
cccagcccca gcgacggaag caccagacag agcagccccg agcctaactc ccccagcgaa 420
tactgcttct atgtggacag cgacatggtg aacgagttca agctggagct ggtggaaaaa 480
ctgttcgccg aggacacaga agccaaaaac cccttcagca cccaggacac agacctggac 540
ctggagatgc tggcccccta catccccatg gacgacgact tccagctgag atccttcgac 600
cagctgagcc ccctggaaag cagcagcgcc tcccccgaat cagccagccc ccagagcacc 660
gtgaccgtgt tccagctgaa aaagatcctg aagatcgagg agctggacga gcgggaactg 720
atcgacatcg aagtgtccgg aaaccacctg ttctacgcca acgacatcct gacacacaat 780
agcagcagca gcgacgtgag agtgaaattc agcagaagcg ccgacgcccc cgcctaccag 840
cagggacaga atcagctgta caacgaactg aacctgggca gaagggagga atacgacgtg 900
ctggacaaga ggagaggaag ggaccccgag atgggaggaa aaccacagag aagaaagaac 960
ccacaggagg gactgtacaa cgagctgcag aaggacaaga tggccgaggc ctacagtgaa 1020
attggcatga agggagagag aagaagagga aagggacacg acggcctgta ccagggcctg 1080
agcaccgcta ccaaggacac atacgacgcc ctgcacatgc aggccctgcc accaagatga 1140

Claims (32)

1. A conditionally controlled spliceable chimeric antigen receptor molecule comprising an antigen recognition unit and a signal transduction unit, wherein the antigen recognition unit comprises an antigen recognition domain, a transmembrane domain, a costimulatory signal domain, an N-terminal splice domain, and a degradant; the signal transduction unit comprises a conditional signal response domain, a C-terminal splice domain, and a signaling domain;
wherein the antigen to which the antigen recognition domain binds is selected from one or more of CD47, AXL, EGFR, CD7, CD24, FAP, CD147, HER2, ROR1, ROR2, CD133, ephA2, CD171, CEA, epCAM, TAG, IL-13 ra, EGFRvIII, GD2, fra, PSCA, PSMA, GPC3, CAIX, claudin18.2, VEGFR2, PD-L1, MSLN, MUC1, c-Met, B7-H3, or TROP2 antigen;
the transmembrane domain is selected from one or more of the CD3 zeta, CD4, CD8, CD28 or CD137/4-1BB transmembrane domains;
The costimulatory signaling domain in the antigen-recognition unit is selected from one or more of CD2, CD27, CD28, CD40, OX40, CD137/4-1BB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11 or Dap10 costimulatory signaling domains;
the N-terminal splicing domain is protein intron Int N ；
The degradation agent is salmonella type III secretion system effector protein (SopE) or mutant estrogen receptor (ERm);
the conditional signal response domain is an oxygen dependent degradation domain (ODD);
the C-terminal splicing domain is protein intron Int C ；
The signaling domain is selected from one or more of CD3 ζ, fcyriii, fceri, fc receptor signaling domain, or an Immunoreceptor Tyrosine Activation Motif (ITAM) -bearing signaling molecule.
2. The chimeric antigen receptor molecule according to claim 1, wherein the antigen to which the antigen recognition domain binds is a CD47 antigen.
3. The chimeric antigen receptor molecule according to claim 1, wherein the transmembrane domain is a CD28 transmembrane domain.
4. The chimeric antigen receptor molecule according to claim 1, wherein the costimulatory signaling domain in the antigen recognition unit is selected from one or more of the CD28 or CD137/4-1BB costimulatory signaling domains.
5. A chimeric antigen receptor molecule according to claim 1, wherein the signaling domain is a CD3 ζ signaling domain.
6. The chimeric antigen receptor molecule according to claim 1, wherein the signal transduction unit further comprises a costimulatory signal domain.
7. The chimeric antigen receptor molecule according to claim 6, wherein the costimulatory signaling domain in the signaling unit is selected from one or more of CD2, CD27, CD28, CD40, OX40, CD137/4-1BB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, or Dap10 costimulatory signaling domain.
8. The chimeric antigen receptor molecule according to any one of claims 1 to 7, wherein the antigen recognition unit has an amino acid sequence as set forth in SEQ NO:1 or SEQ NO:2 is shown in the figure; the amino acid sequence of the signal transduction unit is shown in SEQ NO: 3.
9. A nucleic acid molecule encoding the chimeric antigen receptor molecule of any one of claims 1 to 8.
10. The nucleic acid molecule of claim 9, wherein in the nucleic acid molecule the nucleotide sequence encoding the signal transduction unit further comprises a nucleotide sequence encoding a conditional signal response element.
11. The nucleic acid molecule of claim 9, wherein the condition signal responsive element is selected from one or more of a Hypoxia Responsive Element (HRE), a temperature responsive element, a pH responsive element, a light sensitive element, or an inflammatory factor responsive element.
12. The nucleic acid molecule of claim 9, wherein the condition signal responsive element is a Hypoxia Responsive Element (HRE).
13. The nucleic acid molecule of claim 9, wherein the nucleotide sequence encoding the signal transduction unit is set forth in SEQ NO: 4.
14. The nucleic acid molecule of any one of claims 9 to 13, wherein the nucleotide sequence of the nucleic acid molecule is set forth in SEQ NO:5 or 6.
15. A vector comprising the nucleic acid molecule of any one of claims 9 to 14.
16. The vector of claim 15, wherein the vector is selected from one or more of a plasmid, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a herpes simplex viral vector, a forest encephalitis viral vector, a polio viral vector, a newcastle disease viral vector, or a transposon.
17. The vector of claim 15, wherein the vector is a lentiviral vector.
18. A genetically engineered host cell comprising an exogenous nucleic acid molecule according to any one of claims 9 to 14 integrated in its chromosome, or comprising a vector according to any one of claims 15 to 17.
19. The host cell of claim 18, wherein the host cell is selected from one or more of an isolated human cell or a genetically engineered immune cell.
20. The host cell of claim 19, wherein the isolated human-derived cells are selected from one or more of embryonic stem cells, cord blood-derived stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, T cells, NK cells, or macrophages.
21. The host cell of claim 19, wherein the isolated human cell is a NKT cell.
22. The host cell of claim 19, wherein the genetically engineered immune cell is selected from one or more of a genetically engineered T cell, NK cell, or macrophage.
23. The host cell of claim 19, wherein the genetically engineered immune cell is a NKT cell.
24. The host cell of claim 22, wherein the genetically engineered immune cell is a genetically engineered T cell.
25. The host cell of claim 19, wherein the genetically engineered immune cell is selected from one or more of a chimeric antigen receptor T cell, a chimeric antigen receptor NK cell, a chimeric antigen receptor macrophage, or a T cell receptor T cell.
26. The host cell of claim 19, wherein the genetically engineered immune cell chimeric antigen receptor NKT cell.
27. The host cell of claim 19, wherein the genetically engineered immune cell is a chimeric antigen receptor T cell.
28. A method of preparing a genetically engineered host cell according to any one of claims 18 to 26, comprising introducing a nucleic acid molecule according to any one of claims 9 to 14 and/or a vector according to any one of claims 15 to 17 into a host cell.
29. The method of claim 28, wherein the introducing is transfection or transduction.
30. Use of a chimeric antigen receptor molecule according to any one of claims 1 to 8, a nucleic acid molecule according to any one of claims 9 to 14, a vector according to any one of claims 15 to 17 or a genetically engineered host cell according to any one of claims 18 to 27 in the manufacture of a cellular immunotherapeutic medicament, wherein the cellular immunotherapeutic medicament is for the treatment of a solid tumor.
31. The use of claim 30, wherein the solid tumor is selected from one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, renal cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, or brain cancer.
32. The use of claim 30 or 31, wherein the solid tumor is colorectal cancer.
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