CN111925448A - Preparation method of in vivo-generated CAR-macrophage and application of in vivo-generated CAR-macrophage in tumor immunotherapy - Google Patents
Preparation method of in vivo-generated CAR-macrophage and application of in vivo-generated CAR-macrophage in tumor immunotherapy Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and relates to a preparation method of CAR-macrophage generated in vivo and application of CAR-macrophage generated in tumor immunotherapy. The CAR-macrophage prepared by the method can thoroughly eliminate malignant tumors, and is expected to become a novel immunotherapy tool for treating malignant tumors.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a chimeric antigen receptor and a preparation method thereof, an expression cassette of the chimeric antigen receptor and a preparation method thereof, a nano-carrier containing the chimeric antigen receptor expression cassette, a chimeric antigen receptor-macrophage, a preparation method of the chimeric antigen receptor-macrophage (CAR-macrophage) synthesized in vitro or generated in vivo, and application of the chimeric antigen receptor-macrophage (CAR-macrophage) in tumor immunotherapy.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The clinical effect of chimeric antigen receptor (CAR-T) modified T cell therapy in immunotherapy is significant, and the method is one of the focuses and hot spots of cancer immunotherapy research. Clinical practice proves that when CAR-T plays an anti-tumor effect, a large amount of cytokines are released, and the double-edged sword is played: on one hand, the tumor cells are destroyed by secreted granular enzymes with cytotoxic effect, perforin and the like, and on the other hand, potential safety hazards exist due to cytokine storm: CAR-T cell therapy can cause T cells, B cells, NK cells, etc. to release large amounts of inflammatory cytokines such as IL-6, TNF-alpha, IFN-gamma, etc., triggering acute inflammatory responses to induce epithelial and tissue damage, leading to microvascular leakage, causing nausea, headache, tachycardia, tachypnea, and even other fatal clinical responses.
As an important innate immune cell, macrophages are the first line of defense against infection in the body and are involved in the regulation of various immune-related diseases. Unlike cytotoxic effects that are dominated by T cell "secretion", macrophages primarily play a role in "endocytosis", digestion, and antigen presentation. And the macrophages can compete for oxygen and nutrients better than T cells, and the activity is stronger. Therefore, if the tumor cells are specifically phagocytized by 'domestication' M phi and the antigen presentation characteristic is 'mobilized', the 'endocytic' M phi is expected to become a novel immunotherapy tool for treating malignant tumors.
Disclosure of Invention
In view of the above-mentioned deficiencies of the prior art, the present disclosure for the first time develops a method for in vivo generation of chimeric antigen receptor-macrophages (CAR-macrophages), which can specifically target malignant tumors and enhance the immunotherapy effect on malignant tumors.
The invention is realized by the following technical scheme:
in a first aspect of the invention, a chimeric antigen receptor is provided that includes an extracellular domain, a transmembrane region, and an intracellular signaling domain.
In a second aspect of the invention, there is provided an expression cassette for a chimeric antigen receptor, the gene encoding the chimeric antigen receptor being located on a vector; preferably, the vector is constructed to form a CAR plasmid expression vector containing a promoter that specifically edits macrophages to avoid off-target gene editing, a gene encoding the extracellular domain of a Chimeric Antigen Receptor (CAR), a gene encoding the transmembrane domain of a Chimeric Antigen Receptor (CAR), a gene encoding the intracellular signaling domain of a Chimeric Antigen Receptor (CAR), GFP-luc.
In a third aspect of the invention, a method is provided for in vivo production of chimeric antigen receptor-macrophages (CAR-macrophages) by in vitro preparation of nanocarriers for the nano-delivery of CAR plasmids to achieve in vivo production of CAR-macrophages, such as by preparation of, but not limited to, polymer PBAE, metal framework ZIF-8, cationic albumin nanoparticles for delivery.
The fourth aspect of the invention provides a pCAR-polymer multifunctional nanoparticle, which is prepared by taking a PBAE (poly (butylene adipate-co-acrylate)) carrier as an example, and the specific construction method is that
(1) Constructing a CAR plasmid expression vector containing a CD68 promoter, a gene encoding the extracellular domain of a Chimeric Antigen Receptor (CAR), a gene encoding the transmembrane domain of a Chimeric Antigen Receptor (CAR), a gene encoding the intracellular signaling domain of a Chimeric Antigen Receptor (CAR), GFP-luc, constructing a plasmid fragment encoding an overactive form of a transposase comprising a CD68 promoter, iPB7 transposase;
(2) synthesizing a PBAE framework and modifying the amino at the tail end of the framework by adopting a Michael addition reaction to obtain a PBAE polymer with an amino end-blocked;
(3) modifying the PBAE polymer with the end capped by amino group by polypeptide (MTAS-NLS) containing microtubule-associated sequence (MTAS) and Nuclear Localization Signaling (NLS) to obtain PBAE multifunctional polymer nanoparticles with the end capped by amino group; the mixing ratio of the pCAR and the polypeptide modified polymer PBAE (pCAR nano-particle) is selected from 1:35, 1:30, 1:25, 1:20, 1:15 and 1: 10;
(4) mixing pCAR and polypeptide modified PBAE (pCAR nano-particle) according to a certain proportion to obtain the pCAR-polymer multifunctional nano-particle.
In the fifth aspect of the invention, the chimeric antigen receptor-macrophage is constructed and formed based on the chimeric antigen receptor or the expression cassette thereof or the pCAR-polymer multifunctional nanoparticle.
The sixth aspect of the invention provides a preparation method of the chimeric antigen receptor or the expression cassette thereof or the pCAR-polymer multifunctional nanoparticle.
In a seventh aspect of the present invention, there is provided a method for in vivo production of a chimeric antigen receptor-macrophage.
The eighth aspect of the invention provides an application of the chimeric antigen receptor or the expression cassette thereof or the pCAR-polymer multifunctional nanoparticle in preparing in-vivo specificity editing macrophage products or tumor immunotherapy products.
And provides the application of the chimeric antigen receptor-macrophage in preparing tumor immunotherapy products.
The invention has the beneficial effects that:
(1) the invention provides a method for generating chimeric antigen receptor-macrophage (CAR-macrophage) in vivo, which is realized by preparing a nano-carrier in vitro for nano-delivery of CAR plasmid so as to realize the generation of CAR-macrophage in vivo, such as but not limited to one prepared by preparing PBAE polymer, ZIF-8 metal framework and cationic albumin nano-particles.
(2) The present disclosure provides a method for generating chimeric antigen receptor-macrophage (CAR-macrophage) in vivo, which can be used for specifically editing macrophage in vivo, and can express different specific chimeric antigen receptors according to changing the extracellular domain of CAR to target and treat malignant chronic diseases such as GBM and leukemia.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is the structure of transposase plasmid DNA and CAR plasmid DNA of example 1 of the present application;
FIG. 2 is a schematic diagram of the intelligent combination of pCAR-polymer multifunctional nanoparticles of example 2 of the present application;
FIG. 3 is the effect of the mass ratio of PBAE polymer to CAR plasmid and the surface coating of the nucleopore-penetrating polypeptide on nanoparticle size of example 2 of the present application;
FIG. 4 is the effect of the mass ratio of PBAE polymer to CAR plasmid and the surface coating of the nucleopore polypeptide on nanoparticle surface charge of example 2 of the present application;
FIG. 5 is the results of gel electrophoresis experiments of nanoparticles obtained by compressing CAR plasmid with amino-terminated PBAE polymers of example 2 of the present application;
FIG. 6 shows the results of cell viability assays at various time points after in vitro gene transfection of macrophages according to example 3 of the present application;
FIG. 7 is the flow cytometry quantification of Beads that the CAR-macrophages of example 3 of the present application can specifically phagocytose G422 cell membrane;
FIG. 8 is a graph of tumor bioluminescence quantitation in mice of example 4 of the present application.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In view of the problem of potential safety hazard of the chimeric antigen receptor modified T cell therapy in clinical immunotherapy, the disclosure provides a preparation method of in vivo-generated chimeric antigen receptor-macrophage (CAR-macrophage) and application of the in vivo-generated chimeric antigen receptor-macrophage (CAR-macrophage) in tumor immunotherapy.
In a particularly advantageous embodiment of the present invention, the CAR plasmid expression vector constructed in the present invention can express different specific chimeric antigen receptors to target different diseases, such as malignant refractory diseases, e.g., malignant brain glioma GBM, leukemia, etc., by changing the extracellular domain of the CAR.
According to the invention, in order to avoid the gene editing off-target problem, the selective editing of macrophages can be realized by specifically finding out the CD68 promoter, so that the 'off-target risk' is avoided.
In order to improve the targeting of the plasmid pCAR-polymer nanoparticle to a cell nucleus, a polypeptide (MTAS-NLS) containing a microtubule-associated sequence (MTAS) and a Nuclear Localization Signal (NLS) is used for modifying an amino-terminated PBAE polymer, so that the plasmid DNA is promoted to rapidly enter the nucleus through the microtubule.
In order to encapsulate a plasmid fragment which encodes an overactive form of transposase (iPB7) in a nanoparticle, piggyBac transposase mediates the encapsulation, and a target gene fragment is effectively integrated into a chromosome of macrophage through a shearing and pasting mechanism, so that the continuous and stable expression of CAR gene is realized.
According to the invention, the nanoparticles can be prepared from the group consisting of polymer PBAE, metal framework ZIF-8, cationic albumin nanoparticles.
Because of the complex microenvironment in the organism, the microenvironment at the tumor tissue cells is different from the microenvironment in other normal tissues, and under the influence, the immune cells in different microenvironments have differentiation and function difference. Therefore, in the development process of immunotherapy, the results of in vitro cell experiments are often quite different from the in vivo implementation situation, and it is unexpected for those skilled in the art whether an immunotherapy can be effective under the complex environmental conditions in vivo, and whether to edit specific immune cells in vitro to obtain immune cells with certain functions does not mean to specifically edit specific immune cells in vivo and generate immune cells with certain functions, and it is unknown whether the immune cells with certain functions can achieve effective immunotherapy functions in vivo tumor tissue cells.
Through an alternative nanoparticle delivery mode known by a person skilled in the art, the CAR plasmid expression vector and the plasmid containing transposase provided by the invention are delivered into an organism, and the constructed CAR is successfully absorbed and expressed by macrophages under a complex microenvironment in the organism, so that the macrophages are specifically edited in the organism, and the macrophages have a tumor immunotherapy function of targeting tumor cells.
The room temperature refers to indoor environment temperature, and is generally 15-30 ℃.
The organism refers to mammals, such as rabbits, mice, monkeys, sheep, cats, dogs, orangutans, or other animals.
The invention adopts a carrier containing plasmid fragments such as CD68 promoter, gene for coding extracellular domain of Chimeric Antigen Receptor (CAR), CD8 transmembrane region, FcRgamma, PI3k, GFP-luc and the like, and a plasmid containing transposase, can realize specific editing of macrophage in an organism, is not influenced by complex microenvironment in the organism, enables the generation of chimeric antigen receptor-macrophage, and has the tumor immunotherapy function of targeting tumor cells. Compared with CAR-T cells, the chimeric antigen receptor-macrophage disclosed by the invention has many advantages, the CAR-T cells can form cytokine storm, so that potential safety hazards exist on human bodies, in addition, the unique neurotoxicity of CAR-T cell therapy can also seriously harm organisms, and the chimeric antigen receptor-macrophage disclosed by the invention cannot generate toxic or side effects harmful to human bodies and has higher safety. In addition, CAR-macrophages are of widespread origin, such as precursor cells in embryos, hematopoietic stem/progenitor cells, and monocytes.
The chimeric antigen receptor-macrophage generated in vivo or synthesized in vitro can be used as a method for industrially producing the chimeric antigen receptor-macrophage, and the product with the immunologic function is finally obtained by culturing cells or organisms and enriching or extracting the produced chimeric antigen receptor-macrophage.
In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.
Example 1: construction of pCAR plasmid expression vector
Taking brain glioma as an example, the extracellular domain of CAR is designed using the brain glioma-specific marker EGFR as a specific antigen. And (2) designing PCR primers by using plasmid fragments such as a CD68 promoter, anti-EGFR-scFv, a CD8 transmembrane region, FcRgamma, PI3k, GFP-luc and the like, and carrying out PCR amplification (the plasmid fragments such as the CD68 promoter, the CD8 transmembrane region, the FcRgamma, PI3k, GFP-luc and the like adopted by the invention can be inquired in an NCBI database). Carrying out double enzyme digestion on the vector plasmid by adopting endonuclease (such as Not I and Bam H I), mixing double enzyme digestion products according to a proportion, connecting the products under the action of ligase to form an expression plasmid of CAR, screening positive cloning escherichia coli by using a culture medium containing ampicillin, and sequencing and identifying the positive cloning. Meanwhile, fragments containing CD68 promoter, iPB7 transposase and the like are used as templates to construct a transposase plasmid. The structures of transposase plasmid DNA and CAR plasmid DNA are shown in figure 1.
Example 2 preparation and characterization of pCAR-Polymer multifunctional nanoparticles
1. Synthesis of amino terminated PBAE polymers
The synthesis of PBAE skeleton and the amino modification of the end of the skeleton are respectively completed by Michael addition reaction. The method comprises mixing butanediol acrylate and 5-amino-1-pentanol, and stirring at 90 deg.C for 24 hr to obtain acrylate-terminated C32 polymer. C32 and 1- (3-aminopropyl) -4-methylpiperazine were then co-dissolved in tetrahydrofuran and allowed to react for an additional 2 hours at room temperature, the product was precipitated with diethyl ether, dried under vacuum, dissolved in DMSO to make a 100mg/mL solution of C32-122 polymer, and stored at-20 ℃ for further use.
Preparation and characterization of pCAR-polymer multifunctional nanoparticles
Mixing pCAR and polypeptide modified polymer PBAE (pCAR nano-particle) according to the equal proportion of 1:35, 1:30, 1:25, 1:20, 1:15 and 1:10, investigating the efficiency of forming a nano-composite through electrostatic action, and verifying the compression efficiency of the polymer to plasmid through a gel electrophoresis test; and measuring the change condition of the particle size and the potential of the nanoparticles by adopting a DLS method.
Preliminary studies have shown that when the ratio of polymer to plasmid is greater than 10: 1, nanoparticles can be formed, the particle size of the nanoparticles increases with the increase of the proportion of the nanoparticles to the nanoparticles (fig. 3, w/o is nanoparticles without NLS-MTAS polypeptide, w is nanoparticles with NLS-MTAS polypeptide), and the surface is positively charged (fig. 4); after the core-localized polypeptide is coated, the particle size of the nanoparticle is slightly increased (FIG. 3), and the surface charge is inverted to-10.07 mV (FIG. 4). Gel electrophoresis (fig. 5) showed a mass ratio of PBAE polymer to pCAR of 15: 1, can be completely compressed to form stable nanoparticles.
Example 3 biological evaluation
pCAR-polymer multifunctional nanoparticle-mediated macrophage in-vitro transfection experiment
Macrophage 6x10 extracted in vivo4The cells were seeded in 24-well plates and after 24 hours a defined amount of pCAR (5 ug plasmid per well) was added to each well, after 4 hours of incubation the supernatant was aspirated off and 1mL of medium was added to each well and incubated for 48 hours. The results of cell viability assays at different time points after in vitro gene transfection of macrophages are shown in FIG. 6.
2. Phagocytosis specificity assay
In this example, a glioma-specific marker EGFR was used as a specific antigen to design the CAR extracellular domain, and the generated CAR-macrophages were subjected to a specific phagocytosis assay.
Firstly, separating a brain glioma cell membrane and a astrocytoma cell membrane, and coating the fluorescent-labeled silica Beads; the positivity for phagocytosing Beads macrophages was then determined by flow cytometry by co-culture with CAR macrophages. The results showed that 82.17% of macrophages engulfed G422 cell membrane-coated Beads as quantified by flow cytometry; and for the Beads coated by the astrocyte membrane, the CAR-macrophage can not phagocytize basically (the positive rate is 0.72%) (fig. 7), which shows that the CAR-macrophage prepared by the method can specifically phagocytize malignant tumors but not normal cells, and proves the feasibility of the CAR-macrophage prepared by the method for immunotherapy.
EXAMPLE 4 in vivo antitumor study
Malignant brain Glioma (GBM) grows infiltratively, and the existing conventional treatment cannot completely eliminate postoperative residual star-shaped invasion foci, and finally leads to glioma recurrence. This example illustrates post-operative malignant brain gliomas, demonstrating the feasibility of CAR-macrophages prepared according to the present disclosure to completely eliminate malignant tumors.
Fixing a nude mouse by using a brain stereotaxic instrument, quantitatively injecting brain glioma cells to establish an in-situ glioma nude mouse model, inoculating on day 16, excising solid brain tumor tumors by surgery, dividing the surgery mice into 4 groups (n is 3), respectively injecting PBS (a control group), pCAR plasmid, MTAS-NLS modified polymer PBAE and pCAR-polymer multifunctional nanoparticles (taking the cationic compound PBAE prepared by the method for nano delivery of CAR-plasmid as an example to generate CAR-macrophage specifically expressing EGFR receptor in vivo), and evaluating the anti-tumor effect of each group by fluorescence intensity analysis. The fluorescence intensity analysis results are shown in fig. 8, the in vivo fluorescence intensities of the control group, the mice injected with the pCAR plasmid and the mice injected with the MTAS-NLS modified polymer PBAE are continuously increased after 20 days, which indicates that the mice injected with the pCAR plasmid, the mice injected with the pCAR plasmid and the mice injected with the MTAS-NLS modified polymer PBAE all relapse into brain glioma after surgery, but the in vivo fluorescence intensities of the mice injected with the pCAR-polymer multifunctional nanoparticles are continuously reduced, and even completely disappear after 33 days, which indicates that the CAR-macrophage prepared by the present disclosure can completely eliminate malignant brain glioma, and indicates that the CAR-macrophage prepared by the present disclosure can be used for treating malignant tumor.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A chimeric antigen receptor, comprising an extracellular domain, a transmembrane region, an intracellular signaling domain;
preferably, the transmembrane region is CD 8; preferably, the intracellular signaling domain is FcR γ -PI3 k; preferably, the extracellular domain is an antibody prepared with a tumor cell specific marker as a specific antigen, preferably comprising an sc-Fv, Fab, scFab or scIgG antibody fragment; preferably, the extracellular domain is designed with a specific glioma-specific marker EGFR as specific antigen.
2. The method for producing a chimeric antigen receptor according to claim 1, wherein a vector expressing the chimeric antigen receptor is introduced into an organism, and the chimeric antigen receptor is expressed by in vivo-editing macrophages.
3. An expression cassette for a chimeric antigen receptor according to claim 1, wherein the gene encoding the chimeric antigen receptor is located on a vector;
preferably, the vector is a chimeric antigen receptor plasmid;
preferably, a chimeric antigen receptor plasmid expression vector is constructed containing a promoter that specifically edits macrophages to prevent off-target gene editing, a gene encoding the extracellular domain of the Chimeric Antigen Receptor (CAR), GFP-luc; further preferably, the recombinant vector further comprises a gene encoding a transmembrane region of a chimeric antigen receptor, an intracellular signaling domain; preferably, the promoter is a CD68 promoter, preferably, the transmembrane region is CD 8; preferably, the intracellular signaling domain is FcR γ -PI3 k; preferably, the extracellular domain is specific for an antigen which is a tumor cell specific marker, including sc-Fv, Fab, scFab or scIgG antibody fragments; preferably, the extracellular domain is designed with a specific glioma-specific marker EGFR as specific antigen.
4. The method of claim 3, wherein the chimeric antigen receptor is according to claim 1, and the gene encoding the chimeric antigen receptor is located on a vector;
preferably, a CAR plasmid-forming lentiviral expression vector is constructed containing a promoter that specifically edits macrophages to prevent off-target gene editing, a gene encoding the extracellular domain of a Chimeric Antigen Receptor (CAR), GFP-luc; preferably, the recombinant vector further comprises a gene encoding a transmembrane region of a chimeric antigen receptor, an intracellular signaling domain; preferably, the promoter is a CD68 promoter, preferably, the transmembrane region is CD 8; preferably, the intracellular signaling domain is FcR γ -PI3 k; preferably, the extracellular domain is specific for an antigen which is a tumor cell specific marker, including sc-Fv, Fab, scFab or scIgG antibody fragments; preferably, the extracellular domain is designed with a specific glioma-specific marker EGFR as specific antigen.
5. A nano-carrier containing the expression cassette of the chimeric antigen receptor of claim 3, wherein the nano-carrier comprises one or more of a polymer PBAE, a metal framework ZIF-8 and a cationic albumin nano-particle, preferably, the nano-carrier is the polymer PBAE to obtain a pCAR-polymer multifunctional nano-particle, and the specific construction method comprises the following steps of
(1) Constructing a CAR plasmid expression vector containing a CD68 promoter, a gene encoding the extracellular domain of a Chimeric Antigen Receptor (CAR), a gene encoding the transmembrane domain of a Chimeric Antigen Receptor (CAR), a gene encoding the intracellular signaling domain of a Chimeric Antigen Receptor (CAR), GFP-luc, constructing a plasmid fragment encoding an overactive form of a transposase comprising a CD68 promoter, iPB7 transposase;
(2) synthesizing a PBAE framework and modifying the amino at the tail end of the framework by adopting a Michael addition reaction to obtain a PBAE polymer with an amino end-blocked;
(3) modifying the PBAE polymer with the end capped by amino group by polypeptide (MTAS-NLS) containing microtubule-associated sequence (MTAS) and Nuclear Localization Signaling (NLS) to obtain PBAE multifunctional polymer nanoparticles with the end capped by amino group; the mixing ratio of the pCAR and the polypeptide modified polymer PBAE (pCAR nanoparticles) is 1: 10-35, preferably in a ratio selected from 1:35, 1:30, 1:25, 1:20, 1:15, 1: 10;
(4) mixing pCAR and polypeptide modified PBAE (pCAR nano-particle) according to a certain proportion to obtain pCAR-polymer multifunctional nano-particle;
preferably, the synthesis method of the amino-terminated PBAE polymer in the step (2) is as follows, mixing butanediol acrylate and 5-amino-1-pentanol, and stirring for 20-28 hours at 85-100 ℃ to obtain the acrylate-terminated C32 polymer; then, the C32 polymer and 1- (3-aminopropyl) -4-methylpiperazine are co-dissolved in tetrahydrofuran and continue to react for 1-3 hours at room temperature, the product is precipitated by an organic solvent I, dried and dissolved in an organic solvent II to prepare a C32-122 polymer solution for later use,
preferably, the temperature for mixing and stirring the butanediol acrylate and the 5-amino-1-pentanol during the synthesis of the amino-terminated PBAE polymer is 90 ℃ or 24 hours; preferably, the time for C32 to react with 1- (3-aminopropyl) -4-methylpiperazine co-dissolved tetrahydrofuran during the synthesis of the amino terminated PBAE polymer is 2 hours; preferably, an amino-terminated poly (. beta. -aminoester) polymer (PBAE, C32-122) was developed, preferably, organic solvent I comprises diethyl ether, preferably, organic solvent II comprises DMSO, preferably, the C32-122 polymer solution is at a concentration of 100mg/mL or stored at-20 ℃.
6. A chimeric antigen receptor-macrophage, characterized in that it is based on the chimeric antigen receptor-macrophage constructed according to any one of claims 1 to 5.
7. The method of claim 6, wherein the nanocarrier is prepared in vitro for nano-delivery of the chimeric antigen receptor plasmid, and the macrophage is transfected in vitro.
8. A preparation method of in vivo generated chimeric antigen receptor-macrophage is characterized in that a nano-carrier is prepared in vitro for nano-delivery of a chimeric antigen receptor plasmid, and the nano-carrier is delivered to the macrophage transfected in an organism to obtain the chimeric antigen receptor-macrophage according to claim 6, wherein the nano-carrier preferably comprises one or more of a polymer PBAE, a positive metal framework ZIF-8 and a cationic albumin nano-grade.
9. Use of the chimeric antigen receptor of claim 1 or the expression cassette of claim 3 or the nanocarrier of claim 5 or the chimeric antigen receptor-macrophage of claim 6 for the preparation of an in vivo generated chimeric antigen receptor-macrophage product or in an in vivo specifically edited macrophage product or a tumor immunotherapy product.
10. Use of the chimeric antigen receptor-macrophage prepared by the method of claim 7 or 8 in the preparation of a tumor immunotherapeutic product, preferably, the tumor immunotherapeutic product comprises engineered cells.
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