CN111925448A - Preparation method of in vivo-generated CAR-macrophage and application of in vivo-generated CAR-macrophage in tumor immunotherapy - Google Patents

Abstract

Translated fromChinese

本发明属于生物医药技术领域，涉及在体生成CAR‑巨噬细胞的制备方法及肿瘤免疫治疗中的应用，其制备方法为:首先在体外构建pCAR质粒表达载体，然后构建pCAR‑聚合物多功能纳米粒，进行纳米递送CAR质粒以实现在体内生成CAR‑巨噬细胞。本公开制备的CAR‑巨噬细胞能够彻底清除恶性肿瘤，有望成为治疗恶性肿瘤的新型免疫治疗工具。

The invention belongs to the technical field of biomedicine, and relates to a preparation method for in vivo generation of CAR-macrophages and application in tumor immunotherapy. Nanoparticles, perform nano-delivery of CAR plasmids to generate CAR-macrophages in vivo. The CAR-macrophages prepared in the present disclosure can completely remove malignant tumors, and are expected to become a new immunotherapy tool for the treatment of malignant tumors.

Description

Translated fromChinese

在体生成CAR-巨噬细胞的制备方法及肿瘤免疫治疗中的应用Preparation method of in vivo generated CAR-macrophages and application in tumor immunotherapy

技术领域technical field

本发明属于生物医药技术领域，具体涉及一种嵌合抗原受体及其制备方法、一种嵌合抗原受体的表达盒及其制备方法、一种含有嵌合抗原受体表达盒的纳米载体、一种嵌合抗原受体-巨噬细胞，以及一种体外合成或在体生成嵌合抗原受体-巨噬细胞(CAR-巨噬细胞)的制备方法及在肿瘤免疫治疗中的应用。The invention belongs to the technical field of biomedicine, and in particular relates to a chimeric antigen receptor and a preparation method thereof, an expression cassette of a chimeric antigen receptor and a preparation method thereof, and a nanocarrier containing a chimeric antigen receptor expression cassette , a chimeric antigen receptor-macrophage, and a preparation method for in vitro synthesis or in vivo generation of a chimeric antigen receptor-macrophage (CAR-macrophage) and its application in tumor immunotherapy.

背景技术Background technique

公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

免疫疗法中的嵌合抗原受体修饰的T细胞(chimeric antigen receptor Tcells,CAR-T)疗法临床效果显著，是癌症免疫治疗研究的焦点和热点之一。临床实践证实在CAR-T发挥抗肿瘤效应时，释放大量的细胞因子，发挥着“双刃剑”的作用：一方面依靠分泌的具有胞毒作用的颗粒酶、穿孔素等摧毁肿瘤细胞，另一方面也因细胞因子“风暴”存在安全隐患：CAR-T细胞疗法可引起T细胞、B细胞、NK细胞等释放大量的炎性细胞因子如IL-6、TNF-α和IFN-γ等，促发急性炎症反应诱导上皮及组织损伤，导致微血管渗漏，引起恶心、头痛、心动过速、呼吸急促，甚至其他可致死性临床反应。The chimeric antigen receptor-modified T cells (CAR-T) therapy in immunotherapy has remarkable clinical effect and is one of the focuses and hot spots of cancer immunotherapy research. Clinical practice has confirmed that when CAR-T exerts its anti-tumor effect, it releases a large number of cytokines, which play a "double-edged sword" role: on the one hand, it relies on the secreted cytotoxic granzyme and perforin to destroy tumor cells, and on the other hand, it destroys tumor cells. On the one hand, there are also potential safety hazards due to the cytokine "storm": CAR-T cell therapy can cause T cells, B cells, NK cells, etc. to release a large amount of inflammatory cytokines such as IL-6, TNF-α and IFN-γ, etc. Promoting an acute inflammatory response induces epithelial and tissue damage, leading to microvascular leakage, causing nausea, headache, tachycardia, tachypnea, and even other potentially lethal clinical reactions.

作为重要的固有免疫细胞，巨噬细胞是机体抗感染的第一道防线，参与不同免疫相关疾病的调节。与T细胞“分泌”为主的胞毒作用不同，巨噬细胞主要起到“胞吞”、消化和抗原递呈作用。而且巨噬细胞比T细胞能够更好地争夺氧气和营养物质，活力更强。因此，若能“驯化”MΦ特异性吞噬肿瘤细胞，“调动”其抗原递呈特性，则“胞吞型”MΦ有望成为治疗恶性肿瘤的新型免疫治疗工具。As important innate immune cells, macrophages are the body's first line of defense against infection and are involved in the regulation of different immune-related diseases. Different from the cytotoxic role of T cells, which is mainly "secreted", macrophages mainly play the role of "endocytosis", digestion and antigen presentation. And macrophages can compete for oxygen and nutrients better than T cells and are more active. Therefore, if MΦ-specific phagocytosis of tumor cells can be "acclimated" and its antigen-presenting properties can be "mobilized", then "endocytotic" MΦ is expected to become a new immunotherapy tool for the treatment of malignant tumors.

发明内容SUMMARY OF THE INVENTION

针对上述现有技术的不足，本公开首次开发了一种在体生成嵌合抗原受体-巨噬细胞(CAR-巨噬细胞)的方法，能够特异性靶向恶性肿瘤，增强对恶性肿瘤免疫治疗的效果。In view of the above-mentioned deficiencies of the prior art, the present disclosure develops for the first time a method for in vivo generation of chimeric antigen receptor-macrophages (CAR-macrophages), which can specifically target malignant tumors and enhance immunity to malignant tumors. effect of treatment.

本发明是通过如下技术方案实现的：The present invention is achieved through the following technical solutions:

本发明的第一个方面，提供一种嵌合抗原受体，所述嵌合抗原受体包括胞外结构域、跨膜区、胞内信号传导结构域。The first aspect of the present invention provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane region, and an intracellular signaling domain.

本发明的第二个方面，提供一种嵌合抗原受体的表达盒，编码所述嵌合抗原受体的基因位于载体上；优选地，构建形成CAR质粒表达载体，载体含有特异性编辑巨噬细胞避免基因编辑脱靶的启动子、编码嵌合抗原受体(CAR)胞外结构域的基因、编码嵌合抗原受体(CAR)跨膜区结构域的基因、编码嵌合抗原受体(CAR)胞内信号传导结构域的基因、GFP-luc。The second aspect of the present invention provides a chimeric antigen receptor expression cassette, and the gene encoding the chimeric antigen receptor is located on a vector; preferably, a CAR plasmid expression vector is constructed and formed, and the vector contains a specific editing macrophage Phage avoids gene editing off-target promoters, genes encoding chimeric antigen receptor (CAR) extracellular domains, genes encoding chimeric antigen receptor (CAR) transmembrane domains, genes encoding chimeric antigen receptors (CAR) CAR) gene for intracellular signaling domain, GFP-luc.

本发明的第三个方面，提供一种在体生成嵌合抗原受体-巨噬细胞(CAR-巨噬细胞)的方法，通过在体外制备纳米载体用于纳米递送CAR质粒以实现在体生成CAR-巨噬细胞，例如通过制备聚合物PBAE、金属框架ZIF-8、阳离子白蛋白纳米粒进行递送但不限于某一种。A third aspect of the present invention provides a method for in vivo generation of chimeric antigen receptor-macrophages (CAR-macrophages) by preparing nanocarriers in vitro for nano-delivery of CAR plasmids to achieve in vivo generation CAR-macrophages, such as by preparing polymer PBAE, metal framework ZIF-8, cationic albumin nanoparticles for delivery but not limited to a certain one.

本发明的第四个方面，提供一种pCAR-聚合物多功能纳米粒，以聚合物PBAE载体为例制备多功能纳米粒，具体构建方法为In a fourth aspect of the present invention, a pCAR-polymer multifunctional nanoparticle is provided, and the multifunctional nanoparticle is prepared by taking the polymer PBAE carrier as an example. The specific construction method is as follows:

(1)构建含有CD68启动子、编码嵌合抗原受体(CAR)胞外结构域的基因、编码嵌合抗原受体(CAR)跨膜区结构域的基因、编码嵌合抗原受体(CAR)胞内信号传导结构域的基因、GFP-luc的CAR质粒表达载体，构建含CD68启动子，iPB7转座酶的编码转座酶的过度活性形式的质粒片段；(1) Construct a gene containing the CD68 promoter, a gene encoding the extracellular domain of a chimeric antigen receptor (CAR), a gene encoding a transmembrane domain of a chimeric antigen receptor (CAR), and a gene encoding a chimeric antigen receptor (CAR) transmembrane domain. ) The gene of the intracellular signal transduction domain, the CAR plasmid expression vector of GFP-luc, construct the plasmid fragment containing CD68 promoter, the overactive form of the encoding transposase of iPB7 transposase;

(2)采用Michael加成反应分别完成PBAE骨架的合成和骨架末端的氨基修饰，获得氨基封端的PBAE聚合物；(2) The synthesis of the PBAE skeleton and the amino modification of the skeleton end are respectively completed by Michael addition reaction to obtain an amino-terminated PBAE polymer;

(3)用含有微管相关序列(microtubule-associated sequence，MTAS)和核定位信号(nuclear localization signaling，NLS)的多肽(MTAS-NLS)对氨基封端的PBAE聚合物进行修饰，获得氨基封端的PBAE多功能聚合物纳米粒；pCAR与多肽修饰的聚合物PBAE(pCAR纳米粒)混合的比例选自1:35，1:30，1:25，1:20，1:15，1:10；(3) The amino-terminated PBAE polymer was modified with a polypeptide (MTAS-NLS) containing microtubule-associated sequence (MTAS) and nuclear localization signaling (NLS) to obtain amino-terminated PBAE Multifunctional polymer nanoparticles; the mixing ratio of pCAR and polypeptide-modified polymer PBAE (pCAR nanoparticles) is selected from 1:35, 1:30, 1:25, 1:20, 1:15, 1:10;

(4)将pCAR与多肽修饰的聚合物PBAE(pCAR纳米粒)按一定比例混合，获得pCAR-聚合物多功能纳米粒。(4) Mix pCAR with polypeptide-modified polymer PBAE (pCAR nanoparticles) in a certain proportion to obtain pCAR-polymer multifunctional nanoparticles.

本发明的第五个方面，提供一种嵌合抗原受体-巨噬细胞，基于嵌合抗原受体或其表达盒或pCAR-聚合物多功能纳米粒构建形成。The fifth aspect of the present invention provides a chimeric antigen receptor-macrophage, which is constructed and formed based on the chimeric antigen receptor or its expression cassette or pCAR-polymer multifunctional nanoparticles.

本发明的第六个方面，提供一种嵌合抗原受体或其表达盒或pCAR-聚合物多功能纳米粒的制备方法。The sixth aspect of the present invention provides a method for preparing a chimeric antigen receptor or its expression cassette or pCAR-polymer multifunctional nanoparticle.

本发明的第七个方面，提供一种嵌合抗原受体-巨噬细胞的在体生成方法。A seventh aspect of the present invention provides a method for in vivo generation of chimeric antigen receptor-macrophage cells.

本发明的第八个方面，提供一种嵌合抗原受体或其表达盒或pCAR-聚合物多功能纳米粒在制备在体特异性编辑巨噬细胞产品或肿瘤免疫治疗产品中的应用。The eighth aspect of the present invention provides the application of a chimeric antigen receptor or its expression cassette or pCAR-polymer multifunctional nanoparticle in the preparation of an in vivo specific editing macrophage product or a tumor immunotherapy product.

以及提供嵌合抗原受体-巨噬细胞在制备肿瘤免疫治疗产品中的应用。And provide the application of chimeric antigen receptor-macrophage in the preparation of tumor immunotherapy products.

本发明有益效果：Beneficial effects of the present invention:

(1)本发明提供一种在体生成嵌合抗原受体-巨噬细胞(CAR-巨噬细胞)方法，通过在体外制备纳米载体用于纳米递送CAR质粒以实现在体生成CAR-巨噬细胞，例如通过制备聚合物PBAE、金属框架ZIF-8、阳离子白蛋白纳米粒进行递送但不限于某一种。(1) The present invention provides a method for in vivo generation of chimeric antigen receptor-macrophages (CAR-macrophages), by preparing nanocarriers in vitro for nano-delivery of CAR plasmids to achieve in vivo generation of CAR-macrophages Cells, such as but not limited to one, are delivered by preparing polymer PBAE, metal framework ZIF-8, cationic albumin nanoparticles.

(2)本公开提供了一种在体生成嵌合抗原受体-巨噬细胞(CAR-巨噬细胞)的方法，可用于在体特异性编辑巨噬细胞，可根据改变CAR的细胞外结构域，表达不同的特异性嵌合抗原受体来靶向治疗GBM、白血病等恶性顽疾。(2) The present disclosure provides a method for in vivo generation of chimeric antigen receptor-macrophages (CAR-macrophages), which can be used to specifically edit macrophages in vivo, which can be changed according to the extracellular structure of CAR It expresses different specific chimeric antigen receptors to target the treatment of malignant and intractable diseases such as GBM and leukemia.

附图说明Description of drawings

构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。The accompanying drawings forming a part of the present invention are used to provide further understanding of the present invention, and the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute an improper limitation of the present invention.

图1是本申请实施例1的转座酶质粒DNA和CAR质粒DNA的结构；Fig. 1 is the structure of the transposase plasmid DNA and CAR plasmid DNA of theembodiment 1 of the present application;

图2是本申请实施例2的pCAR-聚合物多功能纳米粒的智能组合示意图；2 is a schematic diagram of the intelligent combination of the pCAR-polymer multifunctional nanoparticles of Example 2 of the present application;

图3是本申请实施例2的PBAE聚合物与CAR质粒质量比及穿核孔多肽的表面包覆对纳米粒粒径的影响；Fig. 3 is the effect of the mass ratio of PBAE polymer and CAR plasmid and the surface coating of penetrating pore polypeptide on the particle size of nanoparticles of Example 2 of the present application;

图4是本申请实施例2的PBAE聚合物与CAR质粒质量比及穿核孔多肽的表面包覆对纳米粒表面电荷的影响；Fig. 4 is the effect of the mass ratio of PBAE polymer and CAR plasmid and the surface coating of the nucleopore polypeptide on the surface charge of the nanoparticle of Example 2 of the present application;

图5是本申请实施例2的氨基封端的PBAE聚合物压缩CAR质粒而得的纳米粒子的凝胶电泳实验结果；Fig. 5 is the gel electrophoresis experiment result of the nanoparticle that the amino-terminated PBAE polymer of the embodiment 2 of the present application compresses the CAR plasmid and obtains;

图6是本申请实施例3的巨噬细胞体外基因转染后不同时间点的细胞活力测定结果；6 is the cell viability assay results at different time points after the in vitro gene transfection of macrophages in Example 3 of the present application;

图7是本申请实施例3的CAR-巨噬细胞可特异性吞噬G422细胞膜包覆的Beads的流式细胞术定量结果；Fig. 7 is the flow cytometry quantitative result that the CAR-macrophages of Example 3 of the present application can specifically phagocytose G422 cell membrane-coated Beads;

图8是本申请实施例4的小鼠体内肿瘤生物发光定量图。FIG. 8 is a quantitative diagram of tumor bioluminescence in vivo in mice in Example 4 of the present application.

具体实施方式Detailed ways

应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。It should be noted that the following detailed description is exemplary and intended to provide further explanation of the application. Unless otherwise defined, 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.

需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本申请的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof.

鉴于临床免疫疗法中的嵌合抗原受体修饰的T细胞疗法存在安全隐患的问题，本公开提出了一种在体生成嵌合抗原受体-巨噬细胞(CAR-巨噬细胞)的制备方法及肿瘤免疫治疗中的应用。In view of the potential safety problems of chimeric antigen receptor-modified T cell therapy in clinical immunotherapy, the present disclosure proposes a preparation method for in vivo generation of chimeric antigen receptor-macrophages (CAR-macrophages). and application in tumor immunotherapy.

本发明的一个特别优秀的实施方案中构建的CAR质粒表达载体中嵌合编码抗原受体(CAR)胞外段结构的基因，可根据改变CAR的细胞外结构域，表达不同的特异性嵌合抗原受体来靶向治疗不同的疾病，如恶性脑胶质瘤GBM、白血病等恶性顽疾。In a particularly excellent embodiment of the present invention, the CAR plasmid expression vector constructed by the chimeric gene encoding the extracellular segment of the antigen receptor (CAR) can express different specific chimeras according to the change of the extracellular domain of the CAR. Antigen receptors can be used to target and treat different diseases, such as malignant brain glioma GBM, leukemia and other malignant and intractable diseases.

根据本发明，为规避基因编辑脱靶问题，特异性寻找到CD68启动子可实现巨噬细胞的选择性编辑，从而规避了“脱靶风险”。According to the present invention, in order to avoid the off-target problem of gene editing, the selective editing of macrophages can be achieved by specifically finding the CD68 promoter, thereby avoiding the "off-target risk".

为提高质粒pCAR-聚合物纳米粒对细胞核的靶向性，用含有微管相关序列(microtubule-associated sequence，MTAS)和核定位信号(nuclearlocalizationsignaling，NLS)的多肽(MTAS-NLS)对氨基封端的PBAE聚合物进行修饰，促进质粒DNA经微管快速入核。In order to improve the targeting of plasmid pCAR-polymer nanoparticles to the nucleus, a polypeptide containing microtubule-associated sequence (MTAS) and nuclear localization signaling (NLS) (MTAS-NLS) was used to target the amino-terminated DNA. The PBAE polymer is modified to facilitate the rapid entry of plasmid DNA into the nucleus through microtubules.

为将编码转座酶(iPB7)的过度活性形式的质粒片段同时包封于纳米粒中，由piggyBac转座酶介导，通过剪切和粘贴机制有效地将目的基因片段整合到巨噬细胞的染色体中，实现了CAR基因的持续稳定表达。In order to simultaneously encapsulate the plasmid fragment encoding the overactive form of the transposase (iPB7) into nanoparticles, the piggyBac transposase mediated by the cut-and-paste mechanism effectively integrates the target gene fragment into macrophages. In the chromosome, the continuous and stable expression of the CAR gene is achieved.

根据本发明，纳米粒的制备可选自聚合物PBAE、金属框架ZIF-8、阳离子白蛋白纳米粒。According to the present invention, the preparation of nanoparticles can be selected from polymer PBAE, metal framework ZIF-8, cationic albumin nanoparticles.

由于生物体内存在复杂的微环境，肿瘤组织细胞处的微环境与其他正常组织中的微环境不同，受此影响下，不同微环境下的免疫细胞存在分化和功能的差异。因此免疫疗法的开发过程中，体外细胞实验的结果往往与体内实施情况大相径庭，本领域技术人员无法预料，在生物体内复杂的环境条件下，一种免疫疗法是否能够奏效，体外编辑特定的免疫细胞，获得具有一定功能的免疫细胞并不意味着在体内能实现特异性编辑特定免疫细胞，并生成具有一定功能的免疫细胞，且未知该一定功能的免疫细胞能否在体内肿瘤组织细胞处实现有效的免疫治疗功能。Due to the complex microenvironment in organisms, the microenvironment of tumor tissue cells is different from that in other normal tissues. Under this influence, immune cells in different microenvironments have differences in differentiation and function. Therefore, in the development process of immunotherapy, the results of in vitro cell experiments are often very different from those implemented in vivo, and those skilled in the art cannot predict whether an immunotherapy can work under the complex environmental conditions in vivo, editing specific immune cells in vitro , Obtaining immune cells with certain functions does not mean that specific immune cells can be specifically edited in vivo to generate immune cells with certain functions, and it is unknown whether the immune cells with certain functions can be effective in tumor tissue cells in vivo. immunotherapy function.

通过本领域技术人员可以知晓的可替换的纳米粒递送方式，将本发明提供的CAR质粒表达载体和含有转座酶的质粒，递送至生物体内，在生物体内复杂的微环境下，使所构建的CAR成功被巨噬细胞吸收并表达，实现在生物体内特异性编辑巨噬细胞，使巨噬细胞具有靶向肿瘤细胞的肿瘤免疫治疗功能。The CAR plasmid expression vector and the transposase-containing plasmid provided by the present invention are delivered to the organism through alternative nanoparticle delivery methods known to those skilled in the art, and in the complex microenvironment of the organism, the constructed The CAR is successfully absorbed and expressed by macrophages, which can specifically edit macrophages in vivo, so that macrophages have the function of tumor immunotherapy targeting tumor cells.

本发明所述室温是指室内的环境温度，一般为15～30℃。The room temperature in the present invention refers to the indoor ambient temperature, which is generally 15-30°C.

生物指哺乳类动物，如兔子、鼠、猴子、羊、猫、狗、猩猩或其他动物。Organisms refer to mammals such as rabbits, rats, monkeys, sheep, cats, dogs, orangutans or other animals.

本发明采用含CD68启动子、编码嵌合抗原受体(CAR)胞外结构域的基因、CD8跨膜区、FcRγ、PI3k、GFP-luc等质粒片段的载体和含有转座酶的质粒，可实现在生物体内特异性编辑巨噬细胞，不受生物体内复杂的微环境的影响，使生成嵌合抗原受体-巨噬细胞，且具有靶向肿瘤细胞的肿瘤免疫治疗功能。本公开发明的嵌合抗原受体-巨噬细胞与CAR-T细胞相比具有诸多优势，CAR-T细胞能够形成细胞因子“风暴”对人体存在安全隐患，另外CAR-T细胞疗法的独特神经毒性也能严重危害机体，本公开发明的嵌合抗原受体-巨噬细胞不会产生危害人体的毒副作用，具有较高的安全性。此外，CAR-巨噬细胞来源广泛，如胚胎中的前体细胞、造血干细胞/祖细胞以及单核细胞。The present invention adopts a vector containing a CD68 promoter, a gene encoding an extracellular domain of a chimeric antigen receptor (CAR), a CD8 transmembrane region, FcRγ, PI3k, GFP-luc and other plasmid fragments and a plasmid containing a transposase. To achieve specific editing of macrophages in vivo, without being affected by the complex microenvironment in the organism, to generate chimeric antigen receptor-macrophages, and to have tumor immunotherapy functions targeting tumor cells. Compared with CAR-T cells, the chimeric antigen receptor-macrophages disclosed in this disclosure have many advantages. The ability of CAR-T cells to form a cytokine "storm" poses a safety hazard to the human body. In addition, the unique neurological characteristics of CAR-T cell therapy Toxicity can also seriously endanger the body, and the chimeric antigen receptor-macrophage disclosed in the present disclosure will not produce toxic and side effects that endanger the body, and has high safety. In addition, CAR-macrophages come from a wide range of sources, such as precursor cells in embryos, hematopoietic stem/progenitor cells, and monocytes.

在体生成或者体外合成嵌合抗原受体-巨噬细胞都可以作为产业上生产嵌合抗原受体-巨噬细胞的方法，通过培育细胞或生物，富集或提取生产得到的嵌合抗原受体-巨噬细胞，最终得到具有免疫功能的产品。In vivo production or in vitro synthesis of chimeric antigen receptor-macrophages can be used as a method for industrial production of chimeric antigen receptor-macrophages. By culturing cells or organisms, enrichment or extraction of the produced chimeric antigen receptors Body-macrophages, and finally get products with immune function.

为了使得本领域技术人员能够更加清楚地了解本公开的技术方案，以下将结合具体的实施例与对比例详细说明本公开的技术方案。In order to enable those skilled in the art to understand the technical solutions of the present disclosure more clearly, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments and comparative examples.

实施例1：pCAR质粒表达载体的构建Example 1: Construction of pCAR plasmid expression vector

以脑胶质瘤为例，用脑胶质瘤特异性标识物EGFR作为特异性抗原设计CAR细胞外结构域。以CD68启动子、anti-EGFR-scFv、CD8跨膜区、FcRγ、PI3k、GFP-luc等质粒片段，设计PCR引物并进行PCR扩增(本发明采用的CD68启动子、CD8跨膜区、FcRγ、PI3k、GFP-luc等质粒片段均能在NCBI数据库查询到)。采用内切酶(如NotⅠ和Bam HⅠ)将载体质粒进行双酶切，并将双酶切产物按照比例混合，在连接酶作用下连接形成CAR的表达质粒，用含有氨苄霉素的培养基筛选阳性克隆大肠杆菌，并将阳性克隆进行测序鉴定。同时以含CD68启动子，iPB7转座酶等片段为模板构建转座酶的质粒。转座酶质粒DNA和CAR质粒DNA的结构如图1所示。Taking glioma as an example, the extracellular domain of CAR was designed using the glioma-specific marker EGFR as the specific antigen. With plasmid fragments such as CD68 promoter, anti-EGFR-scFv, CD8 transmembrane region, FcRγ, PI3k, GFP-luc, etc., design PCR primers and carry out PCR amplification (CD68 promoter, CD8 transmembrane region, FcRγ used in the present invention) , PI3k, GFP-luc and other plasmid fragments can be queried in the NCBI database). Use endonucleases (such as NotI and Bam HI) to double-enzyme digestion of the vector plasmid, and mix the double-enzyme digestion products according to the proportion, and ligase to form a CAR expression plasmid under the action of ligase, and screen with medium containing ampicillin Positive clone E. coli, and the positive clone was identified by sequencing. At the same time, a plasmid containing CD68 promoter, iPB7 transposase and other fragments was used as a template to construct a transposase plasmid. The structures of the transposase plasmid DNA and the CAR plasmid DNA are shown in Figure 1.

实施例2pCAR-聚合物多功能纳米粒的制备及表征Example 2 Preparation and characterization of pCAR-polymer multifunctional nanoparticles

1.氨基封端的PBAE聚合物的合成1. Synthesis of amino-terminated PBAE polymers

采用Michael加成反应分别完成PBAE骨架的合成和骨架末端的氨基修饰。方法如下，将丁二醇丙烯酸酯和5-氨基-1-戊醇混合，在90℃下搅拌24小时即得丙烯酸酯封端的C32聚合物。随后将C32和1-(3-氨基丙基)-4-甲基哌嗪共溶四氢呋喃室温继续反应2小时，产物经乙醚沉淀，真空干燥后，溶于DMSO制成100mg/mL的C32-122聚合物溶液，-20℃低温保存备用。The synthesis of the PBAE backbone and the amino modification of the end of the backbone were accomplished by Michael addition reaction, respectively. The method is as follows, mixing butanediol acrylate and 5-amino-1-pentanol, and stirring at 90° C. for 24 hours to obtain an acrylate-terminated C32 polymer. Subsequently, C32 and 1-(3-aminopropyl)-4-methylpiperazine were co-dissolved in tetrahydrofuran and the reaction was continued for 2 hours at room temperature. The product was precipitated with ether, dried in vacuo, and dissolved in DMSO to prepare 100 mg/mL of C32-122 The polymer solution was stored at -20°C for later use.

2.pCAR-聚合物多功能纳米粒的制备及表征2. Preparation and characterization of pCAR-polymer multifunctional nanoparticles

将pCAR与多肽修饰的聚合物PBAE(pCAR纳米粒)按1:35，1:30，1:25，1:20，1:15，1:10等比例混合，考察通过静电作用形成纳米复合物的效率，并通过凝胶电泳试验验证聚合物对质粒的压缩效率；采用DLS法测定纳米粒粒径和电位的变化情况。Mix pCAR and polypeptide-modified polymer PBAE (pCAR nanoparticles) in equal proportions of 1:35, 1:30, 1:25, 1:20, 1:15, 1:10 to investigate the formation of nanocomplexes by electrostatic interaction And the compression efficiency of the polymer to the plasmid was verified by gel electrophoresis test; the change of nanoparticle size and potential was measured by DLS method.

处方初步研究发现，当聚合物与质粒的比例大于10：1的时候可形成纳米粒，并且随着两者比例的增大纳米粒子的粒径呈现增大趋势(图3，w/o为不含有NLS-MTAS多肽的纳米粒，w为含有NLS-MTAS多肽的纳米粒)，表面带正电(图4)；包覆核定位多肽后纳米粒粒径略增大(图3)，表面电荷翻转为-10.07mV(图4)。凝胶电泳(图5)显示出PBAE聚合物与pCAR质量比在15：1时可完全压缩形成稳定纳米粒子。Preliminary research on the formulation found that nanoparticles can be formed when the ratio of polymer to plasmid is greater than 10:1, and as the ratio of the two increases, the particle size of the nanoparticles tends to increase (Figure 3, w/o is no. Nanoparticles containing NLS-MTAS polypeptide, w is the nanoparticle containing NLS-MTAS polypeptide), the surface is positively charged (Fig. 4); the particle size of the nanoparticles increases slightly after coating with nuclear localization polypeptide (Fig. 3), and the surface charge flipped to -10.07mV (Figure 4). Gel electrophoresis (Fig. 5) showed that the PBAE polymer to pCAR mass ratio of 15:1 could fully compress to form stable nanoparticles.

实施例3生物学评价Example 3 Biological evaluation

1.pCAR-聚合物多功能纳米粒介导的巨噬细胞体外转染实验1. In vitro transfection of macrophages mediated by pCAR-polymer multifunctional nanoparticles

体内提取的巨噬细胞6x10⁴/well种于24孔板中，24小时后，每孔加入定量的pCAR(质粒每孔5ug)，培养4小时后，吸走上清，每孔加入1mL培养基，培养48小时。巨噬细胞体外基因转染后不同时间点的细胞活力测定结果如图6所示。The macrophages extracted in vivo were seeded in a 24-well plate at 6x10⁴ /well. After 24 hours, quantitative pCAR (plasmid 5ug per well) was added to each well. After 4 hours of culture, the supernatant was aspirated and 1 mL of medium was added to each well. , cultured for 48 hours. The cell viability assay results of macrophages at different time points after gene transfection in vitro are shown in Figure 6.

2.吞噬特异性测定2. Phagocytosis Specificity Assay

本实施例以脑胶质瘤为例，用脑胶质瘤特异性标识物EGFR作为特异性抗原设计CAR细胞外结构域，对生成的CAR-巨噬细胞进行特异性吞噬测定。In this example, glioma is used as an example, and the glioma-specific marker EGFR is used as the specific antigen to design the CAR extracellular domain, and the generated CAR-macrophages are specifically phagocytosed.

首先分离脑胶质瘤细胞膜和星型胶质细胞膜，并对荧光标记的二氧化硅Beads进行包被；然后通过与CAR巨噬细胞共同培养，通过流式细胞仪测定吞噬Beads巨噬细胞阳性率。结果显示通过流式细胞仪定量结果显示有82.17％的巨噬细胞吞噬了G422细胞膜包覆的Beads；而对星型胶质细胞膜包覆的Beads，CAR-巨噬细胞基本不会吞噬(阳性率＝0.72％)(图7)，表明了本公开制备的CAR-巨噬细胞能够特异性吞噬恶性肿瘤，而不会吞噬正常细胞，证明了本公开制备的CAR-巨噬细胞用于免疫治疗的可行性。First, the glioma cell membrane and astrocyte membrane were separated and coated with fluorescently labeled silica Beads; then by co-culturing with CAR macrophages, the positive rate of phagocytic Beads macrophages was determined by flow cytometry . The results showed that 82.17% of the macrophages phagocytosed the G422 membrane-coated Beads by flow cytometry; while for the astrocyte membrane-coated Beads, CAR-macrophages basically did not phagocytose (positive rate). = 0.72%) (Fig. 7), indicating that the CAR-macrophages prepared in the present disclosure can specifically phagocytose malignant tumors, but not normal cells, which proves that the CAR-macrophages prepared in the present disclosure can be used for immunotherapy. feasibility.

实验例4体内抗肿瘤研究Experimental Example 4 In vivo anti-tumor study

恶性脑胶质瘤(GBM)呈浸润性生长，目前常规治疗无法彻底清除术后残存星形侵袭灶，最终导致胶质瘤复发。本实例以术后恶性脑胶质瘤为例，验证本公开制备的CAR-巨噬细胞彻底清除恶性肿瘤的可行性。Malignant glioma (GBM) grows invasively, and the current conventional treatment cannot completely remove the residual astrocytic invasive foci after operation, which eventually leads to the recurrence of glioma. This example takes postoperative malignant glioma as an example to verify the feasibility of the CAR-macrophages prepared in the present disclosure to completely remove malignant tumors.

以脑立体定位仪固定裸鼠，定量注射脑胶质瘤细胞建立原位胶质瘤裸鼠模型，接种第16天，通过手术将脑肿瘤实体瘤切除，将手术小鼠分为4组(n＝3)，分别注射PBS(对照组)、pCAR质粒、MTAS-NLS修饰的聚合物PBAE、pCAR-聚合物多功能纳米粒(以本公开制备的阳离子化合物PBAE进行纳米递送CAR-质粒为例在体生成特异性表达EGFR受体的CAR-巨噬细胞)，并通过荧光强度分析评价各组的抗肿瘤作用。荧光强度分析结果如图8所示，对照组、注射pCAR质粒以及注射MTAS-NLS修饰的聚合物PBAE的小鼠均在20天之后体内荧光强度不断升高，表明对照组、注射pCAR质粒以及注射MTAS-NLS修饰的聚合物PBAE小鼠均术后脑胶质瘤复发，但注射过pCAR-聚合物多功能纳米粒小鼠体内荧光强度不断降低，甚至在第33天完全消失，说明本公开制备的CAR-巨噬细胞能够彻底清除恶性脑胶质瘤，表明本公开制备的CAR-巨噬细胞能够用于治疗恶性肿瘤。The nude mice were fixed with a brain stereotaxic apparatus, and glioma cells were quantitatively injected to establish an in situ glioma nude mouse model. On the 16th day of inoculation, the solid brain tumors were surgically removed, and the surgical mice were divided into 4 groups (n =3), inject PBS (control group), pCAR plasmid, MTAS-NLS-modified polymer PBAE, and pCAR-polymer multifunctional nanoparticles (taking the cationic compound PBAE prepared in the present disclosure for nano-delivery of CAR-plasmid as an example) In vivo generation of CAR-macrophages specifically expressing EGFR receptors), and the antitumor effect of each group was evaluated by fluorescence intensity analysis. The results of fluorescence intensity analysis are shown in Figure 8. The fluorescence intensity in the control group, the mice injected with pCAR plasmid and the mice injected with MTAS-NLS-modified polymer PBAE all increased continuously after 20 days, indicating that the control group, the mice injected with pCAR plasmid and injected with The MTAS-NLS-modified polymer PBAE mice all had postoperative brain glioma recurrence, but the fluorescence intensity in the mice injected with pCAR-polymer multifunctional nanoparticles continued to decrease, and even disappeared completely on the 33rd day, indicating that the preparation of the present disclosure The CAR-macrophages can completely remove malignant gliomas, indicating that the CAR-macrophages prepared in the present disclosure can be used to treat malignant tumors.

应注意的是，以上实例仅用于说明本发明的技术方案而非对其进行限制。尽管参照所给出的实例对本发明进行了详细说明，但是本领域的普通技术人员可根据需要对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。It should be noted that the above examples are only used to illustrate the technical solutions of the present invention but not to limit them. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or equivalently replace the technical solutions of the present invention as required without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

Translated fromChinese

1.一种嵌合抗原受体，其特征在于，所述嵌合抗原受体包括胞外结构域、跨膜区、胞内信号传导结构域；1. a chimeric antigen receptor, it is characterised in that the chimeric antigen receptor comprises an extracellular domain, a transmembrane region, an intracellular signaling domain;优选地，所述跨膜区为CD8；优选地，所述胞内信号传导结构域为FcRγ-PI3k；优选地，所述胞外结构域为以肿瘤细胞特异性标志物为特异性抗原制备的抗体，优选地，包括sc-Fv、Fab、scFab或scIgG抗体片段；优选地，所述胞外结构域以特异性脑胶质瘤特异性标识物EGFR为特异性抗原设计。Preferably, the transmembrane region is CD8; preferably, the intracellular signaling domain is FcRγ-PI3k; preferably, the extracellular domain is prepared by using tumor cell specific markers as specific antigens Antibodies, preferably, include sc-Fv, Fab, scFab or scIgG antibody fragments; preferably, the extracellular domain is designed with a specific glioma-specific marker, EGFR, as a specific antigen.2.如权利要求1所述的嵌合抗原受体的制备方法，其特征在于，将表达所述嵌合抗原受体的载体导入生物体内，使在体编辑巨噬细胞表达嵌合抗原受体。2 . The method for preparing a chimeric antigen receptor according to claim 1 , wherein a vector expressing the chimeric antigen receptor is introduced into a living body, so that the edited macrophages express the chimeric antigen receptor in vivo. 3 . .3.一种嵌合抗原受体的表达盒，其特征在于，嵌合抗原受体如权利要求1所述，编码所述嵌合抗原受体的基因位于载体上；3. An expression cassette for a chimeric antigen receptor, wherein the chimeric antigen receptor is as claimed in claim 1, and the gene encoding the chimeric antigen receptor is located on a carrier;优选地，所述载体为嵌合抗原受体质粒；Preferably, the vector is a chimeric antigen receptor plasmid;优选地，构建形成嵌合抗原受体质粒表达载体，载体含有特异性编辑巨噬细胞避免基因编辑脱靶的启动子、编码嵌合抗原受体(CAR)胞外结构域的基因、GFP-luc；进一步优选地，还含有编码嵌合抗原受体跨膜区、胞内信号传导结构域的基因；优选地，启动子为CD68启动子，优选地，所述跨膜区为CD8；优选地，所述胞内信号传导结构域为FcRγ-PI3k；优选地，所述胞外结构域以肿瘤细胞特异性标志物为特异性抗原，包括sc-Fv、Fab、scFab或scIgG抗体片段；优选地，所述胞外结构域以特异性脑胶质瘤特异性标识物EGFR为特异性抗原设计。Preferably, a chimeric antigen receptor plasmid expression vector is constructed and formed, and the vector contains a promoter for specifically editing macrophages to avoid off-target gene editing, a gene encoding an extracellular domain of a chimeric antigen receptor (CAR), and GFP-luc; Further preferably, it also contains a gene encoding a chimeric antigen receptor transmembrane region and an intracellular signaling domain; preferably, the promoter is CD68 promoter, preferably, the transmembrane region is CD8; preferably, the The intracellular signaling domain is FcRγ-PI3k; preferably, the extracellular domain uses tumor cell-specific markers as specific antigens, including sc-Fv, Fab, scFab or scIgG antibody fragments; preferably, the The extracellular domain is designed with the specific glioma-specific marker EGFR as the specific antigen.4.如权利要求3所述的表达盒的制备方法，其特征在于，嵌合抗原受体如权利要求1所述，编码所述嵌合抗原受体的基因位于载体上；4. The method for preparing an expression cassette according to claim 3, wherein the chimeric antigen receptor is as claimed in claim 1, and the gene encoding the chimeric antigen receptor is located on a carrier;优选地，构建形成CAR质粒慢病毒表达载体，载体含有特异性编辑巨噬细胞避免基因编辑脱靶的启动子、编码嵌合抗原受体(CAR)胞外结构域的基因、GFP-luc；优选地，还含有编码嵌合抗原受体跨膜区、胞内信号传导结构域的基因；优选地，启动子为CD68启动子，优选地，所述跨膜区为CD8；优选地，所述胞内信号传导结构域为FcRγ-PI3k；优选地，所述胞外结构域以肿瘤细胞特异性标志物为特异性抗原，包括sc-Fv、Fab、scFab或scIgG抗体片段；优选地，所述胞外结构域以特异性脑胶质瘤特异性标识物EGFR为特异性抗原设计。Preferably, a CAR plasmid lentiviral expression vector is constructed, and the vector contains a promoter that specifically edits macrophages to avoid off-target gene editing, a gene encoding an extracellular domain of a chimeric antigen receptor (CAR), and GFP-luc; preferably , also contains a gene encoding a chimeric antigen receptor transmembrane region and an intracellular signaling domain; preferably, the promoter is a CD68 promoter, preferably, the transmembrane region is CD8; preferably, the intracellular The signaling domain is FcRγ-PI3k; preferably, the extracellular domain uses tumor cell-specific markers as specific antigens, including sc-Fv, Fab, scFab or scIgG antibody fragments; preferably, the extracellular domain The domain is designed with the specific glioma-specific marker EGFR as the specific antigen.5.一种含有权利要求3所述嵌合抗原受体的表达盒的纳米载体，其特征在于，纳米载体包括聚合物PBAE、金属框架ZIF-8、阳离子白蛋白纳米粒的一种或多种，优选的，所述纳米载体为聚合物PBAE，得到一种pCAR-聚合物多功能纳米粒，具体构建方法为5. a nanocarrier containing the expression cassette of the chimeric antigen receptor of claim 3, wherein the nanocarrier comprises one or more of polymer PBAE, metal framework ZIF-8, cationic albumin nanoparticles , preferably, the nanocarrier is a polymer PBAE to obtain a pCAR-polymer multifunctional nanoparticle, and the specific construction method is as follows(1)构建含有CD68启动子、编码嵌合抗原受体(CAR)胞外结构域的基因、编码嵌合抗原受体(CAR)跨膜区结构域的基因、编码嵌合抗原受体(CAR)胞内信号传导结构域的基因、GFP-luc的CAR质粒表达载体，构建含CD68启动子，iPB7转座酶的编码转座酶的过度活性形式的质粒片段；(1) Construct a gene containing the CD68 promoter, a gene encoding the extracellular domain of a chimeric antigen receptor (CAR), a gene encoding a transmembrane domain of a chimeric antigen receptor (CAR), and a gene encoding a chimeric antigen receptor (CAR) transmembrane domain. ) The gene of the intracellular signal transduction domain, the CAR plasmid expression vector of GFP-luc, construct the plasmid fragment containing CD68 promoter, the overactive form of the encoding transposase of iPB7 transposase;(2)采用Michael加成反应分别完成PBAE骨架的合成和骨架末端的氨基修饰，获得氨基封端的PBAE聚合物；(2) The synthesis of the PBAE skeleton and the amino modification of the skeleton end are respectively completed by Michael addition reaction to obtain an amino-terminated PBAE polymer;(3)用含有微管相关序列(microtubule-associated sequence，MTAS)和核定位信号(nuclear localization signaling，NLS)的多肽(MTAS-NLS)对氨基封端的PBAE聚合物进行修饰，获得氨基封端的PBAE多功能聚合物纳米粒；pCAR与多肽修饰的聚合物PBAE(pCAR纳米粒)混合的比例为1：10-35，优选地，比例选自1:35，1:30，1:25，1:20，1:15，1:10；(3) The amino-terminated PBAE polymer was modified with a polypeptide (MTAS-NLS) containing microtubule-associated sequence (MTAS) and nuclear localization signaling (NLS) to obtain amino-terminated PBAE Multifunctional polymer nanoparticles; the mixing ratio of pCAR and polypeptide-modified polymer PBAE (pCAR nanoparticles) is 1:10-35, preferably, the ratio is selected from 1:35, 1:30, 1:25, 1: 20, 1:15, 1:10;(4)将pCAR与多肽修饰的聚合物PBAE(pCAR纳米粒)按一定比例混合，获得pCAR-聚合物多功能纳米粒；(4) Mix pCAR with polypeptide-modified polymer PBAE (pCAR nanoparticles) in a certain proportion to obtain pCAR-polymer multifunctional nanoparticles;优选地，步骤(2)中氨基封端的PBAE聚合物合成方法如下，将丁二醇丙烯酸酯和5-氨基-1-戊醇混合，在85～100℃下搅拌20-28小时即得丙烯酸酯封端的C32聚合物；随后将C32聚合物和1-(3-氨基丙基)-4-甲基哌嗪共溶四氢呋喃室温继续反应1～3小时，产物经有机溶剂I沉淀后干燥，溶于有机溶剂II制成C32-122聚合物溶液备用，Preferably, in step (2), the method for synthesizing the amino-terminated PBAE polymer is as follows, mixing butanediol acrylate and 5-amino-1-pentanol, and stirring at 85-100° C. for 20-28 hours to obtain acrylate End-capped C32 polymer; then the C32 polymer and 1-(3-aminopropyl)-4-methylpiperazine are co-dissolved in tetrahydrofuran and continue to react at room temperature for 1 to 3 hours, the product is precipitated in organic solvent I, dried, dissolved in The organic solvent II is made into a C32-122 polymer solution for later use,优选地，氨基封端的PBAE聚合物的合成过程中丁二醇丙烯酸酯和5-氨基-1-戊醇混合搅拌的温度为90℃或搅拌24小时；优选地，氨基封端的PBAE聚合物的合成过程中C32和1-(3-氨基丙基)-4-甲基哌嗪共溶四氢呋喃反应的时间为2小时；优选的，开发氨基封端的聚(β-氨基酯)聚合物(PBAE，C32-122)，优选地，有机溶剂I包括乙醚，优选地，有机溶剂II包括DMSO，优选地，所述C32-122聚合物溶液浓度为100mg/mL或在-20℃下保存。Preferably, the temperature at which butanediol acrylate and 5-amino-1-pentanol are mixed and stirred during the synthesis of the amino-terminated PBAE polymer is 90° C. or stirred for 24 hours; preferably, the synthesis of the amino-terminated PBAE polymer In the process, the reaction time of C32 and 1-(3-aminopropyl)-4-methylpiperazine co-dissolving in tetrahydrofuran is 2 hours; preferably, an amino-terminated poly(β-aminoester) polymer (PBAE, C32 -122), preferably, organic solvent I includes diethyl ether, preferably, organic solvent II includes DMSO, preferably, the concentration of the C32-122 polymer solution is 100 mg/mL or stored at -20°C.6.一种嵌合抗原受体-巨噬细胞，其特征在于，基于权利要求1-5任一项构建的嵌合抗原受体-巨噬细胞。6. A chimeric antigen receptor-macrophage, characterized in that the chimeric antigen receptor-macrophage constructed based on any one of claims 1-5.7.如权利要求6所述的嵌合抗原受体-巨噬细胞的体外合成制备方法，其特征在于，在体外制备纳米载体用于纳米递送嵌合抗原受体质粒，体外转染巨噬细胞即得。7. The in vitro synthesis preparation method of chimeric antigen receptor-macrophages as claimed in claim 6, is characterized in that, in vitro preparation of nanocarriers is used for nanometer delivery of chimeric antigen receptor plasmids, and in vitro transfection of macrophages That's it.8.一种在体生成嵌合抗原受体-巨噬细胞的制备方法，其特征在于，在体外制备纳米载体用于纳米递送嵌合抗原受体质粒，将纳米载体递送至生物体内转染巨噬细胞即得如权利要求6所述的嵌合抗原受体-巨噬细胞，优选地，纳米载体包括聚合物PBAE、阳金属框架ZIF-8、阳离子白蛋白纳米的一种或多种。8. a preparation method of in vivo generation chimeric antigen receptor-macrophage, is characterized in that, preparing nano-carrier in vitro is used for nano-delivery chimeric antigen receptor plasmid, and nano-carrier is delivered to transfection macrophage in vivo. Phagocytosis can obtain the chimeric antigen receptor-macrophage according to claim 6, preferably, the nanocarrier comprises one or more of polymer PBAE, cationic metal framework ZIF-8, and cationic albumin nanometer.9.权利要求1所述的嵌合抗原受体或权利要求3所述的表达盒或权利要求5所述的纳米载体或权利要求6所述的嵌合抗原受体-巨噬细胞在制备在体生成嵌合抗原受体-巨噬细胞产品或在体特异性编辑巨噬细胞产品或肿瘤免疫治疗产品中的应用。9. 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 are prepared in The application of in vivo generation of chimeric antigen receptor-macrophage products or in vivo specific editing of macrophage products or tumor immunotherapy products.10.权利要求7或8所述的方法制备的嵌合抗原受体-巨噬细胞在制备肿瘤免疫治疗产品中的应用，优选的，所述肿瘤免疫治疗产品包括工程化细胞。10. The application of the chimeric antigen receptor-macrophages prepared by the method of claim 7 or 8 in the preparation of tumor immunotherapy products, preferably, the tumor immunotherapy products include engineered cells.

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