An engineered exosome for immunotherapy of pancreatic cancer and its preparation method

Abstract

The present invention provides an engineered exosome for immunotherapy of pancreatic cancer and a preparation method thereof; the CD11b molecule is overexpressed on the surface of macrophages, and the exosomes derived from M1 macrophages are used for engineering transformation to achieve targeted drug delivery to macrophages, connect CD11b antibodies on their surfaces, and load cGAMP molecules and IL-12 mRNA therein, activate the cGAS-STING pathway, stimulate macrophages to produce IFNs, activate effector T cells, and at the same time, polarize M2 macrophages in the tumor microenvironment into M1 macrophages to achieve efficient treatment of pancreatic cancer. The present invention improves the therapeutic effect of exosomes on pancreatic cancer through a series of engineering transformations of exosomes.

Description

Technical Field

[0001] The present invention discloses an engineered exosome for immunotherapy of pancreatic cancer, and also provides a preparation process thereof, belonging to the technical field of biomedical production. Background Art

[0002] Pancreatic cancer has a hidden onset, rapid progression, high mortality, and extremely poor prognosis. It is one of the tumors with the highest known malignancy. Due to the lack of effective screening and diagnosis methods, the vast majority of patients are in the advanced stage at the time of diagnosis and can only receive chemotherapy. Moreover, the pancreatic cancer tumor tissue has sparse blood vessels, and the blood supply is only 1 / 3 of that of normal pancreatic tissue, resulting in the inability to effectively deliver chemotherapy drugs and other active ingredients to the tumor site. Therefore, the median survival time of pancreatic cancer patients is only 5 - 8 months, and the 5-year survival rate is less than 10%.

[0003] Macrophages are derived from bone marrow monocytes and are present in almost all tissues of the body. As an important part of innate immunity, macrophages can quickly respond to pathogens and foreign bodies invading the blood and tissues of the body, trigger an inflammatory response, clear pathogens to maintain the homeostasis of the internal environment of the body, and participate in the non-specific immune response of the body; at the same time, macrophages can also play an antigen-presenting and immune-regulating role in the specific immune response. Due to the good pluripotency and plasticity of macrophages, they can differentiate into different phenotypes and exhibit different functions at different times and in different environments in the body. Under the stimulation of different microenvironmental signals, macrophages mainly differentiate into M1 type and M2 type. The M1 type mainly plays a role in the initial stage of tumors, killing tumor cells by secreting cytokines; with the progression of tumors, M1-type macrophages are gradually domesticated by factors secreted by tumor cells into M2 type, playing an immunosuppressive function and promoting the malignant progression of tumors. Tumor-associated macrophages in the tumor microenvironment mainly exhibit the M2 type.

[0004] The cytoplasmic DNA sensor cyclic GMP-AMP synthase-stimulator of interferon genes signaling pathway (cGAS-STING) is the main source of endogenous type I interferon (IFN) production. The STING signaling pathway can activate macrophages to produce IFNs, which has the potential to initiate or enhance innate and adaptive immune responses and is crucial for the immunotherapy of pancreatic cancer. Free double-stranded DNA in the cytoplasm can activate cGAS, and after binding to double-stranded DNA, cGAS undergoes a conformational change, catalyzing the formation of 2’, 3’-cyclic GMP-AMP (cGAMP) from ATP and GTP. The second messenger cGAMP can activate STING located on the endoplasmic reticulum. Then, STING forms a tetramer through a higher-order oligomerization reaction and is transported from the endoplasmic reticulum to the endoplasmic reticulum-Golgi intermediate compartment. In the Golgi apparatus, the STING tetramer can serve as a signaling platform to recruit and activate the dimer of TANK-binding kinase 1 (TBK1). In turn, TBK1 can phosphorylate the C-terminal domain of STING to recruit interferon regulatory factor 3 (IRF3) for activation. Phosphorylated IRF3 translocates to the nucleus as a transcription factor to promote the transcriptional expression of IFNs, thereby activating effector T cells. However, STING is a cytoplasmic protein, and the cytoplasmic delivery efficiency of its natural agonists such as cGAMP is poor, with low serum stability and rapid tissue clearance, which hinders its therapeutic effect.

[0005] Exosomes are extracellular vesicles with a size of 40 - 160 nm secreted by living cells, and they have the advantages of small size, good biocompatibility, low toxicity, and low immunogenicity. Compared with other carriers such as liposomes and nanoparticles, exosomes can protect the stability of nucleotide drugs and mRNA from in vivo degradation, improve their serum stability, and increase their circulation time. Moreover, exosomes can inherit the characteristics of the parental cells and obtain some components of the parental cells, such as proteins, lipids, and nucleic acids, endowing them with homing effects and the ability to activate immune responses. In addition, exosomes contain transmembrane and membrane-anchored proteins, increasing their engineering ability, thus enhancing the endocytosis of target cells and promoting the delivery of their internal contents. Summary of the Invention

[0006] The present invention constructs an engineered exosome for the immunotherapy of pancreatic cancer, using exosomes derived from M1 macrophages for engineering transformation to achieve targeted drug delivery to macrophages.

[0007] To achieve the purpose of the present invention, the following technical solutions are adopted:

[0008] After culturing M1 macrophages in large quantities, M1 macrophages were electroporated using cell nanoporation technology. After culturing for 24 hours, the culture medium supernatant was centrifuged, and the supernatant was ultrafiltered and concentrated to obtain crude exosomes, which were further purified using an ultracentrifuge to obtain purified exosomes; cGAMP and IL-12 mRNA were loaded into the purified exosomes through cell nanoporation technology, and then CD11b antibody was incubated with the exosomes. The unloaded therapeutic drugs and unmodified CD11b antibody were washed away with PBS to obtain engineered exosomes, that is, exosomes loaded with therapeutic drugs and modified with CD11b antibody.

[0009] A preparation method of an engineered exosome for immunotherapy of pancreatic cancer according to the present invention comprises the following steps:

[0010] 1) Transfer macrophages THP-1 into a cell culture flask, incubate with RMPI1640 containing 0.1 mg / mL PMA overnight, and the cells are transformed from a suspension state to an adherent state; then incubate with Opti MEM medium containing 0.4 mg / mL LPS, and the cells are transformed into M1 macrophages;

[0011] 2) Take the supernatant in step 1) and perform electroporation, centrifugation, filtration, and ultracentrifugation in sequence to obtain purified exosomes;

[0012] Among them, exosomes are extracted from the supernatant using cell nanoporation technology, filtered using a 0.22 μm filter membrane, and the filtrate is centrifuged using an ultracentrifuge at 100000 g;

[0013] 3) Loading of therapeutic drugs and surface modification:

[0014] Load cGAMP and IL-12 mRNA into the purified exosomes in step 2), then add CD11b antibody for incubation. The incubated mixture is centrifuged at 4 °C using an ultrafiltration tube, and then PBS is added for centrifugation and washing to remove the unloaded therapeutic drugs and unmodified CD11b antibody, obtaining engineered exosomes (Exo-aCD11b-cGAMP-IL-12 mRNA).

[0015] The present invention also defines that the targeted therapeutic drug loading adopts cell nanoporation technology, wherein:

[0016] The process of loading drugs by cell nanoperforation technology is as follows: Collect the purified exosomes and attach 500 μL of them with a protein concentration of 2 mg / mL to the surface of the chip with a nanochannel array. Add 500 μL each of cGAMP at a concentration of 100 ng / mL and IL-12 mRNA at 50 ng / mL to the buffer channel. Under an instantaneous pulse of 220 V, load cGAMP and IL-12 mRNA into the purified exosomes to obtain engineered exosomes loaded with drugs (Exo-cGAMP-IL-12 mRNA).

[0017] Furthermore, the present invention also defines the engineered exosomes obtained by the defined method. The exosomes have a disc-like morphology, contain the exosome characteristic protein CD63, and have an average diameter of 100 nm. The engineered exosomes prepared by the present invention can activate the cGAS-STING pathway and activate effector T cells; reverse the tumor microenvironment, polarize M2 macrophages into M1 macrophages, and inhibit the growth of pancreatic cancer.

[0018] The engineered exosomes (Exo-aCD11b-cGAMP-IL-12 mRNA) described in the present invention can be used to prepare drugs for treating pancreatic cancer.

[0019] The positive effects of the present invention are as follows: It provides an engineered exosome that can immunotherapy pancreatic cancer; uses exosomes derived from M1 macrophages for engineering transformation, overexpresses the CD11b molecule on the surface of macrophages, realizes targeted drug delivery to macrophages, connects CD11b antibodies on its surface, and loads cGAMP molecules and IL-12 mRNA therein, activates the cGAS-STING pathway, stimulates macrophages to produce IFNs, activates effector T cells, and at the same time, realizes the polarization of M2 macrophages into M1 macrophages in the tumor microenvironment, achieving efficient treatment of pancreatic cancer. The present invention improves the therapeutic effect of exosomes on pancreatic cancer through a series of engineering transformations of exosomes. Description of the Drawings

[0020] Figure 1 is the Western blot of exosomes of M1 macrophages of the present invention;

[0021] Figure 2 is the cryo-electron microscopy of Exo-aCD11b-cGAMP-IL-12mRNA of the present invention;

[0022] Figure 3 is the flow cytometry analysis of macrophage uptake of Exo-aCD11b of the present invention;

[0023] Figure 4 is the tumor volume change graph of each group of tumor-bearing models of the present invention;

[0024] Figure 5 It is the graph of the body weight changes of each tumor-bearing model of the present invention during the treatment period;

[0025] Figure 6 It is the survival period of each tumor-bearing model of the present invention. Embodiment

[0026] The present invention is 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 achieved by those of ordinary skill in the art will fall within the scope of the claims of the present invention.

[0027] Example 1

[0028] (1)Extraction of exosomes secreted by M1 macrophages: Transfer macrophages THP-1 into a cell culture flask, incubate with RMPI1640 containing 0.1 mg / mL PMA overnight, and the cells transform from a suspended state to an adherent state; then incubate with Opti MEM medium containing 0.4 mg / mL LPS, and the cells transform into M1 macrophages; after culturing for 2 days, digest the cells, centrifuge at 300 g for 5 minutes, collect M1 macrophages, place them on a cell nanoporator chip, and perform electroporation under the conditions of 200-220 v, 5-10 ms, 5-10 pulses, and an interval time of 0.1-0.5 s, and then perform cell culture according to the cell amount of 5-8 million cells per flask for 24 hours to increase the exosome yield;

[0029] (2)Take the supernatant and perform gradient centrifugation at 4 °C using a high-speed refrigerated centrifuge: Centrifuge at 300 g for 10 min to remove dead cells, centrifuge at 1200 g for 10 min, and centrifuge at 10000 g for 20 min to remove cell debris; take the supernatant, filter it with a sterile 0.22 μm filter membrane to remove larger vesicles. Finally, centrifuge at 100000 g for 70 min using an ultracentrifuge to precipitate M1 macrophage exosomes. The obtained M1 macrophage exosomes have exosome marker proteins CD63, TSG101, and Alix on their surface. See Figure 1 ;

[0030] (3)Mix the purified M1 macrophage exosomes with 50-100 μg cGAMP and 50-100 ng IL-12mRNA, and use cell nanoporator technology to add the two drugs into the exosomes. Under the condition of 4 °C, centrifuge at 100000 g for 2 h using an ultracentrifuge to remove the drugs that have not entered the exosomes, and obtain Exo-cGAMP-IL12 mRNA;

[0031] (4)Loading of anti-CD11b antibody on the surface of exosomes: The exosomes obtained by ultracentrifugation were incubated with anti-CD11b at 37 °C for 2 h (protein content 1:1). Under the condition of 4 °C, ultracentrifugation was carried out at 100,000 g for 2 h to remove the antibodies that did not bind to Exo-cGAMP-IL12 mRNA, and Exo-aCD11b-cGAMP-IL12 mRNA was obtained. The obtained Exo-aCD11b-cGAMP-IL12 mRNA was spherical, with a particle size of about 100 nm and good dispersibility. See Figure 2 .

[0032] Take the Exo-aCD11b-cGAMP-IL12 mRNA obtained in Example 1, stain it with PKH26, and then incubate it with THP-1 macrophages. Flow cytometry analysis was performed on the uptake of Exo-aCD11b-cGAMP-IL12 mRNA by THP-1. The specific steps are as follows:

[0033] Add PKH26 dye to 0.3 - 0.5 mg / mL Exo-aCD11b (100 - 120 μL), incubate at room temperature for 20 - 40 min, centrifuge at 1200 g for 20 min at 4 °C using a 100 kDa ultrafiltration tube, add 150 μL PBS, centrifuge at 1200 g for 20 min at 4 °C using a 100 kDa ultrafiltration tube, and repeat once to obtain PKH26-stained exosomes (PKH26-Exo-aCD11b);

[0034] Add PKH26-Exo-aCD11b to THP-1 macrophages, incubate for 4 h, remove the supernatant, wash with PBS, digest with trypsin, centrifuge at 800 g for 5 min, remove the supernatant and add 400 μL paraformaldehyde solution for fixation. Flow cytometry was used to analyze the uptake of PKH26-Exo-aCD11b by THP-1 macrophages. The experimental results showed that THP-1 macrophages had a relatively high uptake of PKH26-Exo-aCD11b; see Figure 3 Flow cytometry analysis chart of macrophage uptake of Exo-aCD11b.

[0035] Construction of pancreatic cancer tumor model: BALB / c nude mice (male, 18 - 20 g, 4 weeks old) were purchased from Liaoning Changsheng Co., Ltd. PANC-02 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. Pentobarbital sodium was intraperitoneally injected at 60 mg / kg. A midline abdominal incision was made, the pancreas was pulled out, and 1×106 After injecting the suspension of hamster pancreatic cancer cells at [X] cells / L, the abdomen was routinely closed.

[0036] In vivo treatment: All tumor-bearing mice were divided into 7 groups, with 5 tumor-bearing mice in each group. On the 10th, 14th, 17th, and 21st days after tumor injection in BALB / c nude mice, 100 μL of Exo-aCD11b-cGAMP-IL12 mRNA (cGAMP 100 μg / mouse, mRNA 0.086 nmol / mouse), 100 μL of Exo-aCD11b-cGAMP (cGAMP 100 μg / mouse), Exo-aCD11b-IL12 mRNA (mRNA 0.086 nmol / mouse), 100 μL of Exo-aCD11b, 100 μL of siRNA (siRNA 0.086 nmol / mouse), 100 μL of cGAMP, and 100 μL of PBS were injected into the tumor-bearing mice via the tail vein. 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 Exo-aCD11b-cGAMP-IL12 mRNA can significantly prolong the survival period of tumor-bearing mice and the body weight change of this group of tumor-bearing mice is not significant; moreover, Exo-aCD11b-cGAMP-IL12 mRNA shows better effects than other single-loaded drugs.

[0037] 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 present invention patent in other related technical fields, are regarded as within the protection scope of the present invention patent. Note: The value in "[X] cells / L" in the translation of line needs to be filled with the specific value in the original text. Also, the specific content of " 6 ", " ", etc. may need to be adjusted according to the actual situation in the patent context, as they might be some kind of internal identifiers or specific notations in the original patent text. Here, they are directly retained as in the original.

Claims

1. An engineered exosome for immunotherapy of pancreatic cancer, characterized in that: The engineered exosome Exo-aCD11b-cGAMP-IL-12 mRNA is disc-shaped, contains the exosome characteristic protein CD63, and has an average diameter of 100 nm; it can activate the cGAS-STING pathway, activate effector T cells; reverse the tumor microenvironment, polarize M2 macrophages into M1 macrophages, and inhibit the growth of pancreatic cancer; The engineered exosome is loaded with therapeutic drugs and surface-modified by the following method: The collected purified exosomes are attached to the surface of the chip of the nanochannel array at a protein concentration of 2 mg / mL and 500 μL. cGAMP at a concentration of 100 ng / mL and IL-12 mRNA at 50 ng / mL, each 500 μL, are added to the buffer channel. Under an instantaneous pulse of 220 V, cGAMP and IL-12mRNA are loaded into the purified exosomes to obtain the drug-loaded engineered exosome Exo-cGAMP-IL-12 mRNA; Finally, CD11b antibody is added for incubation. The incubated mixture is centrifuged at 4°C with an ultrafiltration tube, and PBS is added for centrifugation and washing to remove the unloaded therapeutic drugs and unmodified CD11b antibody, obtaining the engineered exosome Exo-aCD11b-cGAMP-IL-12 mRNA loaded with therapeutic drugs and surface-modified.

2. A preparation method of an engineered exosome for immunotherapy of pancreatic cancer according to claim 1, comprising the following steps: 1) Transfer macrophages THP-1 into a cell culture flask, incubate with RMPI1640 containing 0.1 mg / mL PMA overnight, and the cells are transformed from a suspension state to an adherent state; then incubate with Opti MEM medium containing 0.4 mg / mL LPS, and the cells are transformed into M1 macrophages; 2) Take the supernatant in step 1) and perform electrotransformation, centrifugation, filtration, and ultracentrifugation in sequence to obtain purified exosomes; Among them, the supernatant uses cell nanoporation technology to extract exosomes, the filtration uses a 0.22 μm filter membrane for filtration, and the filtrate is centrifuged with an ultracentrifuge at 100,000 g; 3) Load therapeutic drugs and surface modification: The collected purified exosomes are attached to the surface of the chip of the nanochannel array at a protein concentration of 2 mg / mL and 500 μL. cGAMP at a concentration of 100 ng / mL and IL-12 mRNA at 50 ng / mL, each 500 μL, are added to the buffer channel. Under an instantaneous pulse of 220 V, cGAMP and IL-12mRNA are loaded into the purified exosomes to obtain the drug-loaded engineered exosome Exo-cGAMP-IL-12 mRNA; Finally, add CD11b antibody for incubation. After incubation, the mixture is centrifuged at 4°C using an ultrafiltration tube, and then PBS is added for centrifugation washing to remove unloaded therapeutic drugs and unmodified CD11b antibody, obtaining exosomes Exo-aCD11b-cGAMP-IL-12 mRNA loaded with therapeutic drugs and surface-modified.

3. Use of the engineered exosomes Exo-aCD11b-cGAMP-IL-12 mRNA according to claim 1 in the preparation of a drug for treating pancreatic cancer.

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