Engineered exosome for immunotherapy of lung cancer as well as preparation method and medical application of engineered exosome

Abstract

The invention discloses an engineered exosome for immunotherapy of lung cancer and a preparation method of the engineered exosome, particularly relates to a method for delivering synNotch-CAR mRNA (messenger Ribonucleic Acid) into a body through the exosome for in-situ modification of macrophages, and provides the engineered exosome capable of immunotherapy of lung cancer. The exosome derived from the M1 macrophages is used for engineering transformation, and targeted delivery of drugs to the macrophages is realized. The synNotch-CAR-TAMs in-vivo transformation strategy provided by the invention has high controllability, tissue specificity and durability, is expected to be expanded to various solid tumor types in the future, and provides a precise treatment scheme with higher clinical transformation value for cancer patients. The exosome is subjected to a series of engineering modification, so that the treatment effect of the exosome on lung cancer is improved.

Description

Technical Field

[0001] The present invention discloses an engineered exosome for lung cancer immunotherapy and a preparation method thereof, relating to a gene delivery system based on engineered exosomes and its application in tumor immunotherapy, in particular to a method for in-situ remodeling macrophages in vivo by delivering synNotch-CAR mRNA through exosomes, and is used for preparing drugs for treating lung cancer diseases, belonging to the field of medical technology. Background Art

[0002] In the basic research and clinical applications of current cancer treatment, the targeting strategies of most polymer nanodrugs mainly focus on tumor cells, aiming to directly inhibit their proliferation or induce apoptosis. However, the tumor microenvironment (TME) plays a crucial role in the occurrence, progression, and immune escape of cancer. Among them, tumor-associated macrophages (TAMs), as a key component of the TME, usually exhibit an immunosuppressive M2 phenotype, which can promote angiogenesis, matrix remodeling, and T cell exhaustion, weaken the anti-tumor immune response, and thus lead to the non-responsiveness of approximately 70% of solid tumor patients to immune checkpoint inhibitor (ICI) therapy. Therefore, treatment strategies targeting only tumor cells often fail to achieve durable anti-tumor effects, and regulating the TME and remodeling the immune microenvironment have become the key directions for improving cancer treatment efficacy.

[0003] In recent years, engineered immune cell therapies have made important progress in the field of cancer treatment. Among them, chimeric antigen receptor T cell (CAR-T) therapy has achieved remarkable efficacy in hematological malignancies and has been approved for clinical use. However, in the treatment of solid tumors, CAR-T therapy faces multiple challenges, including immunosuppression in the tumor microenvironment (TME), heterogeneity of solid tumors, tumor antigen loss, and insufficient infiltration and persistence of CAR-T cells. In addition, CAR-T cells mainly rely on the adaptive immune mechanism to exert their functions, and there are a large number of immunosuppressive cells in the solid tumor TME, such as TAMs, which can weaken the efficacy of CAR-T by secreting immunosuppressive factors and promoting T cell exhaustion, resulting in limited application of CAR-T in solid tumors. In view of the limitations of CAR-T therapy in solid tumors, in recent years, CAR macrophages (CAR-M) have gradually attracted attention as a new type of cell therapy. Macrophages can not only directly phagocytose and clear tumor cells, but also further activate the adaptive immune response through antigen presentation function. Therefore, CAR-M is expected to make up for the deficiencies of CAR-T in the treatment of solid tumors. However, the current CAR-M therapy mainly uses in vitro engineering modification, with a limited survival time in vivo and a lack of dynamic adaptability to the TME, making it difficult to maintain the anti-tumor effect in the solid tumor environment for a long time.

[0004] Exosomes are extracellular vesicles with a size of 40-160 nm secreted by living cells, featuring small volume, high biocompatibility, low toxicity, and low immunogenicity. Compared with liposomes and nanoparticles, exosomes can protect nucleotide drugs and mRNA from degradation, improve serum stability, and prolong the circulation time. In addition, exosomes inherit the components of the parental cells and possess homing effects and immune activation capabilities, thus overcoming the TME barrier of solid tumors and in situ transforming TAMs into anti-tumor effector synNotch-CAR-M, which can continuously exert its effects locally in the tumor. Summary of the Invention

[0005] The present invention discloses an engineered exosome for lung cancer immunotherapy and its preparation method. By engineering the exosomes derived from M1 macrophages, synNotch-CAR mRNA is delivered to macrophages to in situ engineer TAMs into synNotch-CAR-M in vivo, which has obvious curative effects on the treatment of lung cancer.

[0006] The engineered exosome for lung cancer immunotherapy according to the present invention is obtained through the following technical solutions: After large-scale culturing of M1 macrophages, they are electrotransfected using cell nanoporation technology. After 24 hours, the supernatant is collected, and crude exosomes are obtained through centrifugation and ultrafiltration, and then purified exosomes are obtained through ultracentrifugation. synNotch-CAR mRNA is loaded into the purified exosomes using cell nanoporation technology to obtain engineered exosomes (synNotch-CAR-exo).

[0007] The engineered exosomes (synNotch-CAR-exo) have a disc-like morphology, contain CD63 exosome characteristic proteins, and have an average diameter of 100 nm. These exosomes can be used to transform M2 macrophages into synNotch-CAR-M in vivo, thereby inducing an adaptive immune response and being effectively used for the treatment of lung cancer.

[0008] The preparation method of the engineered exosome for lung cancer immunotherapy according to the present invention includes the following steps: 1. Polarization of macrophages; After inoculating macrophages for 24 hours, they are incubated with a medium containing 0.1 μg / mL LPS for 48 hours, and the macrophages are polarized into M1 macrophages; 2. Drug loading: Place the M1 macrophages obtained in step 1) on a cell nanoporator chip and perform electroporation (200 - 220 V, 5 - 10 ms, 5 - 10 pulses, pulse interval 0.1 - 0.5 s). Add 50 μL of synNotch-CAR plasmid at a concentration of 100 μg / mL to the buffer channel. Under nanosecond pulses, transfect the synNotch-CAR plasmid into M1 macrophages and culture for 24 hours.

[0009] 3. Purification of exosomes: Collect the supernatant of the cell culture in step 2), extract exosomes, filter using a 0.22 μm filter membrane, and centrifuge the filtrate with an ultracentrifuge at 100,000 g to obtain engineered exosomes (syN-CAR-exo) loaded with drugs.

[0010] The present invention uses exosomes as an engineered gene vector and utilizes the homing effect of exosomes to overcome the TME barrier of solid tumors, in-situ transform TAMs into anti-tumor effector synNotch-CAR-M, and can continuously play a role locally in tumors.

[0011] The engineered exosomes (synNotch-CAR-exo) described in the present invention can be used to prepare drugs for treating lung cancer diseases.

[0012] After engineered TAMs phagocytose tumor cells, on the one hand, they activate CD8⁺ T cells and CD4⁺ Th cells respectively through MHC-I / II molecules, drive the clonal expansion of cytotoxic T cells (CTLs) and the secretion of effector factors such as IFN-γ by Th cells; on the other hand, the activated Th cells differentiate into follicular helper T cells (Tfh), promote B cells to produce tumor-specific antibodies, and then bridge the broad-spectrum killing function of NK cells through antibody-dependent cell-mediated cytotoxicity (ADCC), forming a synergistic effect of "precision clearance of T cells + heterogeneous coverage of NK cells".

[0013] Through the design of the synNotch-CAR system, tumor-associated macrophages (TAMs) can be activated by specific tumor signals in the immunosuppressive microenvironment (TME), thereby continuously releasing pro-inflammatory signals, enhancing the adaptive immune response, promoting the recruitment and activation of effector immune cells such as T cells and NK cells, forming a stronger anti-tumor immune cycle, and achieving efficient treatment of lung cancer.

[0014] In view of the limited efficacy caused by the functional heterogeneity of tumor-associated macrophages (TAMs) and the immunosuppressive microenvironment (TME) in the treatment of solid tumors, a "sensing-activation-synergy" trinity precision regulation strategy is constructed based on the synNotch synthetic biology system: the synNotch receptor recognizes specific signals in the immunosuppressive microenvironment (TME) (such as hypoxia-inducible factor or tumor stromal protease), strictly limiting the activation of CAR only at the tumor site, and using the microenvironment gating mechanism to reduce the off-target risk; at the same time, this signal triggers the dynamic transformation of TAMs from the M2-type immunosuppressive phenotype to the M1-type anti-tumor phenotype, and remodels the immune-supportive microenvironment by secreting factors such as IL-12 and TNF-α. After the engineered TAMs phagocytose tumor cells, on the one hand, they activate CD8⁺ T cells and CD4⁺ Th cells respectively through MHC-I / II molecules, driving the clonal expansion of cytotoxic T cells (CTLs) and the secretion of effector factors such as IFN-γ by Th cells; on the other hand, the activated Th cells differentiate into follicular helper T cells (Tfh), promoting B cells to produce tumor-specific antibodies, and then bridging the broad-spectrum killing function of NK cells through antibody-dependent cell-mediated cytotoxicity (ADCC), forming a synergistic effect of "precision clearance of T cells + heterogeneous coverage of NK cells". By in-situ reprogramming TAMs in vivo, this invention synchronously integrates targeted control, immune microenvironment remodeling and the coordinated functions of multiple immune cell populations, breaking through the bottlenecks such as tissue penetration barriers, off-target toxicity and T cell exhaustion faced by traditional CAR therapies in solid tumors, and providing a new generation of immunotherapy solutions with high safety and strong adaptability for refractory tumors such as lung cancer.

[0015] The positive effects of this invention are as follows: It provides an engineered exosome that can immunotherapy lung cancer; the exosome derived from M1 macrophages is engineered to achieve targeted drug delivery to macrophages. The in-vivo modification strategy of synNotch-CAR-TAMs provided by this invention has high controllability, tissue specificity and persistence, and is expected to be extended to various solid tumor types in the future, providing a more clinically translatable precision treatment plan for cancer patients. By performing a series of engineering modifications on exosomes, this invention improves the therapeutic effect of exosomes on lung cancer.

[0016] Compared with traditional in vitro engineered CAR-M and lipid nanoparticle (LNP) delivery strategies, it has significant innovative advantages. Traditional in vitro engineered CAR-M relies on the isolation, in vitro gene editing and reinfusion of macrophages. It not only has cell activity loss, abnormal activation of phenotypes and bottlenecks in large-scale production, but also leads to functional distortion due to cells being detached from the native TME; while the LNP delivery system can achieve in vivo gene delivery, it is easily cleared rapidly by the mononuclear phagocyte system (MPS), and due to the lack of targeting ligands, it causes non-specific uptake, and combined with low endosomal escape efficiency, it is difficult to achieve efficient transfection of TAMs. The present invention utilizes the natural characteristics of exosomes to break through the above limitations: on the one hand, endogenous proteins on the surface of exosomes (such as milk adhesion factor, integrin) endow it with the ability to actively target TAMs, which can avoid MPS clearance and accurately deliver genes to target cells; on the other hand, its structural characteristics homologous to the cell membrane significantly improve the endosomal escape efficiency, ensuring the efficient intracellular expression of the synNotch-CAR gene, and the low immunogenicity of exosomes avoids triggering systemic toxic reactions. In addition, the tumor antigen-dependent activation characteristics of the synNotch-CAR system enable TAMs to initiate targeted phagocytosis and immune activation functions only when contacting tumor antigens, effectively avoiding off-target toxicity and cell exhaustion caused by constitutive activation of traditional CAR-M. At the same time, exosomes can co-load immunomodulatory factors (such as miRNA, cytokines) to synergistically reshape the immunosuppressive TME, and this multifunctional synergistic effect cannot be achieved by the single metabolic carrier of LNP. This technology integrates the biological advantages of exosomes and the precise regulation of synthetic biology, endows TAMs with targeted therapeutic functions while retaining their in-situ adaptability, and provides a breakthrough solution for lung cancer immunotherapy. Brief Description of the Drawings

[0017] Figure 1 is the Western blot of M1 macrophage exosomes of the present invention; Figure 2 is the cryo-electron microscopy of synNotch-CAR-exo of the present invention; Figure 3 is the flow cytometry analysis of macrophage uptake of synNotch-CAR-exo of the present invention; Figure 4 is the tumor volume change graph of each group of tumor-bearing models of the present invention; Figure 5 is the body weight change graph of each group of tumor-bearing models during treatment of the present invention; Figure 6 is the survival period of each group of tumor-bearing models of the present invention. Detailed Description of the Invention

[0018] The present invention will be further described by the following examples, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change that is easily achievable by those of ordinary skill in the art will fall within the scope of the claims of the present invention. Example 1

[0019] Preparation of RWA 264.7 synNotch-CAR-exo 1. Extraction of exosomes secreted by RAW 264.7 M1 macrophages: Transfer RAW 264.7 macrophages into a cell culture flask, and then incubate the cells with a medium containing 0.1 μg / mL LPS. The cells are transformed into RAW 264.7 M1 macrophages. After culturing for 48 hours, collect RAW 264.7 M1 macrophages and place them on a cell nanoporation chip for electroporation (200 - 220 V, 5 - 10 ms, 5 - 10 pulses, pulse interval 0.1 - 0.5 s). Electroporate 50 - 100 ng of synNotch-CAR mRNA into the cells and culture them for 24 hours at 5 - 8 million cells per flask to increase exosome production.

[0020] 2. At 4°C, perform gradient centrifugation using a high-speed refrigerated centrifuge. First, centrifuge at 300 g for 10 minutes to remove dead cells, then centrifuge at 1200 g for 10 minutes and 10000 g for 20 minutes to remove cell debris. Subsequently, take the supernatant and filter it through a 0.22 μm sterile filter membrane to remove larger vesicles. Finally, ultracentrifuge at 100000 g for 70 minutes to precipitate RAW 264.7 M1 macrophage exosomes, which have marker proteins such as CD63, CD9, TSG101, and Alix. See Figure 1 ; 3. The obtained RAW 264.7 synNotch-CAR-exo is spherical, with a particle size of about 100 nm and good dispersibility. See Figure 2 。 Example 2

[0021] Preparation of THP-1 synNotch-CAR-exo 1. Extraction of exosomes secreted by THP-1 M1 macrophages: THP-1 macrophages were seeded into a cell culture flask, and then a medium containing 0.1 μg / mL PMA was added to polarize the cells into THP-1 M0 macrophages; after culturing for 48 hours, 0.1 μg / mL LPS was added to polarize THP-1 M0 macrophages into THP-1 M1 macrophages; after collecting THP-1 M1 macrophages, they were placed on a cell nanoporator chip for electroporation (200 - 220 V, 5 - 10 ms, 5 - 10 pulses, pulse interval 0.1 - 0.5 s), and 50 - 100 ng of synNotch-CAR mRNA was electroporated into the cells, and the cells were cultured at 5 - 8 million cells per flask for 24 hours to increase exosome production.

[0022] 2. At 4°C, gradient centrifugation was performed using a high-speed refrigerated centrifuge. First, centrifuge at 300 g for 10 minutes to remove dead cells, then centrifuge at 1200 g for 10 minutes and 10000 g for 20 minutes to remove cell debris; subsequently, take the supernatant, filter it through a 0.22 μm sterile filter membrane to remove large vesicles, and finally ultracentrifuge at 100000 g for 70 minutes to precipitate THP-1 M1 macrophage exosomes, which have marker proteins such as CD63, CD9, TSG101, and Alix on their surface, see Figure 1 ; 3. The obtained THP-1 synNotch-CAR-exo is spherical, with a particle size of about 100 nm and good dispersibility, see Figure 2 。

[0023] The positive effects of the present invention are further illustrated by the following experimental examples: Test Example 1 Take the RWA 264.7 synNotch-CAR-exo and THP-1 synNotch-CA-exo obtained in Example 1, stain them with PKH26, and then incubate them with RAW 264.7 and THP-1 macrophages respectively, and perform flow cytometry analysis on the uptake of RWA 264.7 synNotch-CAR-exo and THP-1 synNotch-CA-exo by RAW 264.7 and THP-1 respectively; The specific steps are as follows:

[0024] Add PKH26 dye to 0.3 - 0.5 mg / mL RAW 264.7 synNotch - CAR - exo or THP - 1 synNotch - CA - exo (100 - 120 μL), incubate at room temperature for 20 - 40 min, centrifuge at 4°C and 1200 g for 20 min using a 100 kDa ultrafiltration tube, add 150 μL PBS, centrifuge at 4°C and 1200 g for 20 min using a 100 kDa ultrafiltration tube, and repeat once to obtain PKH67 - stained exosomes (PKH26 - RAW 264.7 synNotch - CAR - exo and PKH26 - THP - 1 synNotch - CAR - exo); Add PKH26 - RAW 264.7 synNotch - CAR - exo and PKH26 - THP - 1 synNotch - CAR - exo to RAW 264.7 and THP - 1 macrophages respectively, incubate for 4 h, collect the cells, fix them with 400 μL paraformaldehyde solution, and analyze the uptake of PKH26 - RAW 264.7 synNotch - CAR - exo and PKH26 - THP - 1 synNotch - CAR - exo by RAW 264.7 and THP - 1 using a flow cytometer. The experimental results show that RAW 264.7 and THP - 1 macrophages both showed a high uptake of PKH26 - RAW 264.7 synNotch - CAR - exo and PKH26 - THP - 1 synNotch - CAR - exo; See Figure 3 Flow cytometry analysis chart of macrophage uptake of synNotch - CAR - exo.

[0025] Experimental Example 2

[0026] Construction of lung cancer tumor model: C57BL / 6 mice (male, 18 - 20 g, 4 weeks old) were purchased from Liaoning Changsheng Co., Ltd. LLC - Luc cells were stably passaged 5 - 6 generations in DMEM culture medium. When the cell proliferation was vigorous, the culture was terminated. A cell suspension was prepared with serum - free DMEM culture medium, and the cell density was adjusted to 1×10 9 cells / L. Inject sodium pentobarbital intraperitoneally at 60 mg / kg, and inject 1×10 6 cells / L of LLC - Luc cell suspension into the lungs.

[0027] In vivo treatment: All tumor-bearing mice were divided into 4 groups, with 6 tumor-bearing mice in each group: PBS, M1-exo, low-dose RAW264.7 synNotch-CAR-exo, and high-dose RAW 264.7 synNotch-CAR-exo. On the 7th, 12th, 17th, 22nd, 27th, 32nd, and 37th days after orthotopic injection of tumors in C57BL / 6 mice, RAW 264.7 synNotch-CAR-exo was injected into the tumor-bearing mice via the tail vein, and the survival period, body weight, and tumor volume of each group of tumor-bearing mice were observed. The results are as Figure 4 , Figure 5 and Figure 6 shown. It can be proved from the results in the figure that RAW 264.7 synNotch-CAR-exo can significantly prolong the survival period of tumor-bearing mice and the body weight of the tumor-bearing mice in this group does not change much.

[0028] Engineered with exosomes derived from M1 macrophages to achieve the delivery of synNotch-CAR mRNA to macrophages for in-situ engineering of TAMs into synNotch-CAR-M in vivo, which has obvious curative effects on the treatment of lung cancer. It has significant innovative advantages compared with traditional in vitro engineered CAR-M and lipid nanoparticle (LNP) delivery strategies. Traditional in vitro engineered CAR-M relies on the isolation, in vitro gene editing and reinfusion of macrophages. It not only has cell activity loss, abnormal activation of phenotypes and bottlenecks in large-scale production, but also leads to functional distortion due to cells being detached from the native TME; while the LNP delivery system can achieve in vivo gene delivery, but it is easily cleared quickly by the mononuclear phagocyte system (MPS), and due to the lack of targeting ligands, it causes non-specific uptake, and the endosomal escape efficiency is low, making it difficult to achieve efficient transfection of TAMs. The present invention utilizes the natural characteristics of exosomes to break through the above limitations: on the one hand, the endogenous proteins (such as milk adhesion, integrin) on the surface of exosomes endow them with the ability to actively target TAMs, which can avoid MPS clearance and accurately deliver genes to target cells; on the other hand, its structural characteristics homologous to the cell membrane significantly improve the endosomal escape efficiency, ensuring the efficient intracellular expression of the synNotch-CAR gene, and the low immunogenicity of exosomes avoids triggering systemic toxic reactions. In addition, the tumor antigen-dependent activation characteristics of the synNotch-CAR system enable TAMs to initiate targeted phagocytosis and immune activation functions only when they contact tumor antigens, effectively avoiding the off-target toxicity and cell exhaustion caused by constitutive activation of traditional CAR-M. At the same time, exosomes can co-load immunomodulatory factors (such as miRNA, cytokines) to synergistically reshape the immunosuppressive TME, and this multifunctional synergistic effect cannot be achieved by the single metabolic carrier of LNP. This technology integrates the biological advantages of exosomes and the precise regulation of synthetic biology to endow TAMs with targeted therapeutic functions while retaining their in-situ adaptability, providing a breakthrough solution for lung cancer immunotherapy.

[0029] The above is only a general description and implementation method of the present invention, and does not limit the patent protection scope of the present invention. Any equivalent changes made according to the description and drawings of the present invention, or directly or indirectly using the patent of the present invention in other related technical fields, are regarded as within the protection scope of the patent of the present invention.

Claims

1. An engineered exosome for lung cancer immunotherapy, characterized in that: After large-scale culturing of M1 macrophages, electrotransfection is performed using cell nanoporization technology. After 24 hours, the supernatant is collected, and crude exosomes are obtained through centrifugation and ultrafiltration. Then, the pure exosomes are obtained through ultracentrifugation purification; synNotch-CAR mRNA is loaded into the purified exosomes through cell nanoporization technology to obtain engineered exosomes; The engineered exosomes are disc-shaped in morphology, contain the exosomal characteristic protein CD63, and have an average diameter of 100 nm; these exosomes can be used to transform M2 macrophages into synNotch-CAR-M in vivo, thereby inducing an adaptive immune response and being effectively used for the treatment of lung cancer.

2. A preparation method of an engineered exosome for lung cancer immunotherapy according to claim 1, comprising the following steps: 1) Polarization of macrophages; After inoculating macrophages for 24 hours, incubate the cells with a medium containing 0.1 μg / mL LPS for 48 hours, and the macrophages are polarized into M1 macrophages; 2) Drug loading: Take the M1 macrophages in step 1) and place them on a cell nanoporization chip for electroperforation (200 - 220 V, 5 - 10 ms, 5 - 10 pulses, pulse interval 0.1 - 0.5 s). Add 50 μL of synNotch-CAR plasmid at a concentration of 100 μg / mL to the buffer channel. Under nanosecond pulses, transfect the synNotch-CAR plasmid into M1 macrophages and culture for 24 hours; 3) Purification of exosomes: Collect the supernatant of the cell culture in step 2), extract exosomes, filter using a 0.22 μm filter membrane, and centrifuge the filtrate with an ultracentrifuge at 100,000 g to obtain the engineered exosomes loaded with drugs.

3. Use of an engineered exosome for lung cancer immunotherapy according to claim 1 in the preparation of a drug for treating lung cancer diseases.

Images