CN114848838A - Pharmaceutical composition containing dendritic cell exosomes and application thereof - Google Patents

Pharmaceutical composition containing dendritic cell exosomes and application thereof Download PDF

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CN114848838A
CN114848838A CN202210642703.9A CN202210642703A CN114848838A CN 114848838 A CN114848838 A CN 114848838A CN 202210642703 A CN202210642703 A CN 202210642703A CN 114848838 A CN114848838 A CN 114848838A
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dendritic cell
antibody
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洪子涵
戴伟亮
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Nanjing Hantaiyu Biotechnology Co ltd
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Abstract

The application relates to a pharmaceutical composition comprising dendritic cell exosomes and application thereof, wherein the exosomes are used as a drug delivery carrier and form a compound with a targeted Her2 specific antibody, the antibody has a nano antibody structure, can be combined with a target antigen with high specificity, improves the therapeutic targeting property, and has simpler structure, thereby facilitating the preparation and purification of the compound; the exosome is a DC cell exosome Dex, and after the DC cell is induced by a tumor marker and an immune factor, the DC cell can be effectively activated to improve the active ingredients of the exosome and the anti-tumor efficacy; the Dex-antibody compound shows synergistic effect in-vivo and in-vitro experiments, can simultaneously improve the treatment effectiveness and the killing targeting property, prolongs the survival period of experimental animals and regulates the expression level of immune protein.

Description

Pharmaceutical composition containing dendritic cell exosomes and application thereof
The technical field is as follows:
the invention belongs to the field of tumor immunotherapy, and particularly provides a pharmaceutical composition containing dendritic cell exosomes and application thereof.
The background art comprises the following steps:
cancer is a malignant disease that seriously jeopardizes people's life health, wherein breast cancer is the most common cancer in women, accounts for one-tenth of the newly diagnosed cancers every year, and is the second most common cause of cancer death in women in the world. At present, routine examination, imaging examination, especially mammography examination and tissue biopsy can be used to diagnose breast cancer, and early diagnosis is crucial to improve patient survival. However, early breast cancer often shows mild symptoms such as lump in breast, change in shape or size of breast, nipple discharge, and mastalgia, and it is difficult to pay sufficient attention to the early breast cancer and delay the treatment. The causes of breast cancer are complex and have been reported to be related to factors including: (1) age: as the female population ages, the age-adjusted incidence of breast cancer continues to increase; (2) sex: most breast cancers occur in women; (3) personal medical history: a history of cancer in one breast increases the likelihood of a primary cancer in the contralateral breast; (4) bad lifestyle or exposure to dangerous goods: smoking, alcohol abuse, or other harmful chemicals may increase the risk of breast cancer; (5) family history and genetic risk factors for breast cancer: the risk of the first-degree relatives of breast cancer patients suffering from breast cancer is 2 to 3 times higher, and research indicates that BRCA1 and BRCA2 are two most important genes causing increased susceptibility to breast cancer; (6) physiological risk factors: the physiological processes of menarche, first birth, menopause, etc. in women also increase the risk of breast cancer.
Through long-term clinical practice research, two basic treatment principles of breast cancer, namely the reduction of the chance of local recurrence and the risk of metastatic spread, are gradually summarized. Surgery can achieve local control of cancer, such as Halsted radical mastectomy, pedicular modified radical surgery, quadrant resection, etc., but surgical treatment not only increases the economic and life burden of patients, but also has difficulty in coping with invasive tumors. Chemotherapy, hormonal therapy and targeted therapy are systemic therapies for the treatment of breast cancer, which have now become the recommended treatment regimen, such as the use of first generation chemotherapy regimens (such as cyclophosphamide, methotrexate and 5-fluorouracil) over a 6 month period that reduce the risk of relapse by 25% over a 10 to 15 year period. However, the above therapy is still difficult to effectively control the development of breast cancer, improve the life cycle of patients and improve the quality of life, and a novel treatment means with strong targeting, low side effect and obvious anti-tumor effect is urgently needed clinically.
Cancer immunotherapy is one of the new methods currently being studied, including but not limited to adoptive T cell therapy (ACT), Dendritic Cell (DC) vaccine therapy, Natural Killer (NK) cell therapy, chimeric antigen receptor T cell (CAR-T) therapy, immune checkpoint inhibitor therapy, etc., which does not use a toxic chemical agent that is harmful to the body, can avoid severe chemotherapy side effects, improve the quality of life of patients, and can achieve the purpose of effectively killing cancer cells.
Among the numerous immunotherapies, DC cell therapy has attracted attention. The DC cell is an innate immune cell, is involved in stress response of the body to tumor and virus, can be used as an Antigen Presenting Cell (APC) and can promote CD8 by major histocompatibility complex molecules class I (MHC I) and class II (MHC II) respectively + And CD4 + The degree of activation of T cells; DC cells also regulate cytokine and chemokine expression levels, regulating inflammation and homing of lymphocytes. DCs have also been widely used as drug delivery vehicles, such as development of anti-tumor vaccines based on DC cells, with Sipuleucel-t (productive Dendreon corporation) being the first DC vaccine therapy approved by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic prostate cancer at 4 months 2010, with hundreds of DC cell-related clinical trials subsequently being deployed in various countries and regions around the world. However, the preparation of anti-tumor vaccines by using DC cells is challenging, the molecular composition of the DC cells is very easy to change, so that the quality control parameters of the vaccines are difficult to grasp, and the abundance of the tumor-associated antigen MHC-ii complexes on the surfaces of the DC cells is low, so that the yield of the vaccines is low and the price of the vaccines is high. The DC-derived exosomes (Dex) seems to be an effective solution for solving the problems related to DC immunotherapy, the Dex has a membrane component of a DC cell, expresses MHC-I, MHC-II and co-stimulatory molecules, has a basic immune stimulatory function of the DC cell, and reports that a TAA-MHCII complex presented by the Dex is 10-100 times more than that of the DC cell, and the Dex membrane component is easy to control, good in stability, easy to store, good in bioavailability and long in-vivo half-life. In addition, Dex can also be used as a drug delivery carrier carrying small molecule chemical drugs or antibody drugs to play a synergistic antitumor role, such as CN112957341B, CN111840528A, CN113292608B and the likeThe exosome is used as a drug delivery carrier and carries small-molecule or large-molecule drugs.
Human epidermal growth factor receptor 2(HER2), designated erbB-2, or proto-oncogene Neu, is a receptor tyrosine protein kinase encoded by the erbB2(HER2) gene on chromosome 17q12, and the HER2 protein forms heterodimers with ligand-binding members of the EGF receptor family, stabilizing ligand binding and enhancing kinase-mediated downstream signaling, including activation of phosphatylinositol-3 kinase and mitogen-activated protein kinase. HER2 expression was detectable on the cell membrane of gastrointestinal, respiratory, reproductive, urinary, skin, breast and placental epithelial cells, most of the breast cancer cases were of the HER2 positive subtype defined by HER2 protein overexpression and/or HER2 gene amplification, HER2 positive breast cancer was considered the most aggressive subtype, and high recurrence rates were observed prior to introduction of anti-HER 2 targeted therapy. Trastuzumab (a humanized monoclonal antibody targeting HER2) added to conventional adjuvant chemotherapy significantly extended disease-free survival (HR 0.60; 95% Confidence Interval (CI) 0.50-0.71, p <0.001) and overall survival (HR 0.66; 95% CI 0.57-0.77, p < 0.00001). In addition to trastuzumab, several other drugs against HER2, such as the monoclonal antibody pertuzumab, the antibody-drug conjugate trastuzumab-emtansine (T-DM1), and tyrosine kinase inhibitors such as lapatinib and lenatinib, have entered clinical treatment, allowing for the sequential administration of targeted combination therapies or non-cross-resistant drugs. However, treatment with HER2 antibody still faces a relatively high recurrence rate, leaving great room for improvement in this targeted therapy.
In the face of the above situation, the invention tries to prepare a DC cell exosome with high anti-tumor activity and enables the DC cell exosome to carry a high-affinity nano antibody targeting HER2, thereby exerting a high-efficiency tumor killing effect, and providing a new research idea for effectively solving the clinical difficulties that DC cells are difficult to store, complicated to prepare, short in-vivo half-life period, poor in curative effect durability of HER2 antibody, easy to relapse and the like.
Disclosure of Invention
In order to solve the technical problems, the invention provides a pharmaceutical composition for treating malignant tumors, which comprises dendritic cell exosomes carrying a targeted HER2 antibody, wherein the exosomes are connected with a targeted HER2 antibody through a phospholipid polyethylene glycol succinimide ester cross-linking agent, the targeted HER2 antibody is a nanobody, and the nanobody comprises CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3.
Dendritic cell exosomes (Dex) comprise a plurality of anti-tumor active ingredients such as DNA, RNA, cytokines, major tissue compatibility objects MHC-I and MHC-II and the like, and the Dendritic cell exosomes (Dex) have been reported to have an inhibiting effect on a plurality of malignant tumors such as breast cancer, prostate cancer, liver cancer, stomach cancer, melanoma, myeloma, lymphoma and leukemia, are widely used for tumor vaccine research, and have been developed over 100 related clinical test researches all over the world at present, however, in the early research, the effect of treating tumors by using Dex alone is not stable, and large differences are displayed in different types of malignant tumors, even different subtypes of tumors of the same tumor, and the effects are possibly related to the complex components and more factors of natural Dex; in addition, when Dex is used alone for treatment, the tumor targeting is poor, and the targeted treatment effect is difficult to play.
In order to solve the difficulties, a nano antibody (nano-anti HER2) which is obtained by early screening and targets HER2 is utilized, the antibody can be combined with a target antigen with high affinity, only has a heavy chain structure, has a simpler molecular structure and is convenient for later modification treatment. The nano-anti HER2 and Dex are connected by using a phospholipid polyethylene glycol succinimidyl ester cross-linking agent to form a Dex-nano-anti HER2 compound, and the Dex is used as a drug delivery carrier of the compound, so that the bioavailability can be improved, the in vivo half-life period can be prolonged, the drug targeting property can be improved, tumor cells can be specifically eliminated, and the systemic anti-tumor effect can be exerted.
Furthermore, the amino acid sequence of the nano antibody is shown as SEQ ID NO. 4.
Further, the preparation step of the dendritic cell exosome comprises the following steps: use of AIM-V Medium at 5% CO 2 Primarily culturing human DC cells in an incubator at 37 ℃ until the degree of cell fusion is reachedAfter 90%, the medium was replaced with fresh medium at 5% CO 2 Culturing in 37 deg.C incubator for 48 hr; collecting the culture medium, and removing free living cells, dead cells and cell debris in the culture medium by adopting a gradient centrifugation method; filtering the obtained solution by using a 0.22 mu m filter membrane to remove large-particle impurities; then, the mixture was subjected to ultracentrifugation at 10000g at 4 ℃ for 60min, the supernatant was discarded, the mixture was washed 3 times with sterile PBS, and the mixture was subjected to ultracentrifugation at 4 ℃ for 90min at 10000g, and the precipitate was collected to obtain dendritic cell exosome particles.
Further, the preparation step of the dendritic cell exosomes comprises the following steps: adding inducer during DC cell culture process, wherein the inducer comprises one or more of carcinoembryonic antigen (CEA), Carbohydrate Antigen (CA)125, IL-12, IL-10, IFN-gamma and TGF-beta.
Further, the inducing agent comprises carcinoembryonic antigen (CEA), Carbohydrate Antigen (CA)125 and IL-12.
In early studies, it was difficult to achieve satisfactory antitumor effects using Dex produced from natural DC cells. This is probably because the natural DC cells are not in an effective activation state, which results in insufficient antitumor substances contained in Dex, affecting the antitumor effect in vitro experiments, and preliminary determination shows that the natural DC cells Dex have low contents of nucleic acids and proteins, which also proves our guess. Therefore, different inducers are adopted to activate DC cells and promote the secretion of antitumor active substances in the invention.
It has been reported that carcinoembryonic antigen (CEA) and Carbohydrate Antigen (CA)125 are important tumor markers of breast cancer, and are closely related to the occurrence and development of breast cancer, and the abnormal change of the protein secretion belongs to a marker event in the tumor microenvironment, so the above substances are used as DC cell induction reagents in this embodiment; it is also reported that cytokines such as IL-12, IL-10, IFN-gamma, TGF-beta and the like can also participate in the activation process of immune cells, induce the differentiation and maturation of the immune cells and stimulate the secretion of exosomes, so that the breast cancer marker and the cytokines are combined to form an inducing reagent in the invention, the Dex secretion is stimulated, and the anti-tumor effect is improved. Experiments prove that the combined induction effect of carcinoembryonic antigen (CEA), Carbohydrate Antigen (CA)125 and IL-12 is the best, and the Dex anti-tumor capability secreted by DC cells is obviously improved after the combination is stimulated.
Further, the step of linking the dendritic cell exosomes to a HER 2-targeting antibody comprises: uniformly mixing phospholipid polyethylene glycol succinimidyl ester (DSPE-PEG-NHS) dissolved in dimethyl sulfoxide as an organic solvent with an HER2 nano antibody according to a ratio of 1:10-10:1, stirring and mixing for 30min in a sterile environment at 4 ℃, then reacting at 4 ℃ overnight, centrifuging at 5000rpm for 10-15min to remove unreacted phospholipid polyethylene glycol succinimidyl ester to obtain a phospholipid polyethylene glycol succinimidyl ester-HER 2 nano antibody compound; dissolving the dendritic cell exosome in sterile phosphate buffer solution to obtain dendritic cell exosome solution, uniformly mixing the compound and the dendritic cell exosome according to the proportion of 1:5-5:1, gently sucking and uniformly mixing at 4 ℃, reacting for 2-4h at 37 ℃, and centrifuging at 4500rpm for 5min to obtain the dendritic cell exosome carrying the targeted HER2 nano antibody.
According to the invention, phospholipid polyethylene glycol succinimidyl ester is selected as a cross-linking agent to connect Dex and a targeted HER2 antibody, the cross-linking agent is a biocompatible cross-linking agent, and not only can the antibody and Dex be effectively connected to form a binary compound, but also the biological activities of the antibody and Dex can be retained to the greatest extent, the damage to a biological molecule and a phospholipid bilayer with a micro-vesicular structure can be prevented, and the anti-tumor effect can be favorably exerted.
Provides the application of the pharmaceutical composition in preparing antitumor drugs.
Further, the tumor is selected from one or more of breast cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, head and neck cancer, pancreatic cancer, lymphoma, leukemia and/or myeloma.
Further, the tumor is selected from breast cancer.
DC cell exosomes have been reported to have broad antitumor effects; the HER2 target is also expressed in a variety of tumor cells including, but not limited to, breast, lung, stomach, colorectal, prostate, head and neck, pancreatic, lymphoma, leukemia and/or myeloma, among others, and HER2 antibodies have been used clinically to treat a variety of malignancies, such as in the journal literature (HER2 heterologous and resistance to anti-cancer)i-HER2 antibody-drug conjugates，Alberto
Figure BDA0003682643630000061
et al, Breast Cancer Res.2020; 22:15.) reports that the HER2 antibody can be used for treating various malignant tumors such as breast cancer, epithelial cancer, gastric cancer, liver cancer and the like, so that the pharmaceutical composition provided by the invention has quite wide application prospect and can treat various tumors.
Advantageous effects
The application provides a pharmaceutical composition and application thereof, wherein the pharmaceutical composition comprises dendritic cell exosomes carrying a targeted HER2 antibody, and has the following advantages:
(1) the nano antibody with a novel amino acid sequence structure is selected, so that the nano antibody can be combined with a target antigen with high specificity, and the treatment targeting property is improved;
(2) the structure of the nano antibody is simpler, and the preparation and purification of the Dex-antibody compound are easy;
(3) the DC cells can effectively stimulate the secretion of anti-tumor active ingredients such as nucleic acid, cell factors, regulatory protein and the like through an inducer activation program, and improve the tumor killing effect;
(4) the Dex-antibody compound shows obvious synergistic effect in-vivo and in-vitro experiments, can simultaneously improve the treatment effectiveness and the killing targeting property, prolongs the life cycle of experimental animals, and regulates the expression level of immune protein.
Drawings
FIG. 1: DC cell exosome Dex electron microscope picture;
FIG. 2: tumor killing ability of Dex under different induction conditions;
FIG. 3: tumor killing ability of Dex antibody complex;
FIG. 4: changes in Fas protein expression levels;
FIG. 5: experimental animals showed changes in tumor volume.
Detailed Description
The following non-limiting examples enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. All the technologies implemented based on the above-mentioned contents of the present invention should fall within the scope of the claims of the present application.
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagent biomaterials, test kits, if not specifically indicated, are commercially available.
Example 1 design and acquisition of HER2 Nanobody
In a previous study, we designed and prepared a nano-anti HER2 antibody targeting HER2 target using phage display technology, as follows: coating the target protein human HER2 protein in an immune container, and performing 3 rounds of phage enrichment screening; carrying out gene sequencing on the phage obtained by screening to obtain sequence information, and designing and synthesizing the screened nano antibody according to the sequence information; cloning the gene sequence of the nano antibody into an expression vector, then introducing the gene sequence into escherichia coli, and carrying out low-temperature induced expression by using IPTG (isopropyl-beta-thiogalactoside); collecting thallus, crushing under high pressure, centrifuging at 10000g and 4 ℃ to obtain crude antibody protein, and then separating and purifying by Ni column and molecular sieve to obtain pure nano antibody.
After ELISA preliminary verification of the affinity with a target antigen, a molecular interaction analysis platform Biacore is utilized to detect the affinity of the nano antibody and human HER2 protein, and a nano-anti HER2 antibody with the affinity meeting the requirement is screened out, wherein K of the nano-anti HER2 antibody is K D The value is 25.64nM and the affinity is at nanomolar level, indicating that it has a higher target antigen binding activity. Through analysis and determination, the nano antibody comprises CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3, and the amino acid sequence of the nano antibody is shown in SEQ ID NO. 4.
Example 2 preparation of DC extracellular exosomes
2.1 Primary DC cell culture
Human DC cells were primary cultured by known methods, and the culture method disclosed in WO2019213550A1 is referred to in the present invention, and the specific steps include: peripheral Blood Mononuclear Cells (PBMC) from healthy volunteers were obtained from lymphocyte isolates and cultured in a 5% CO2 incubator at 37 ℃ for 2-4 hours. Immature Dendritic Cells (iDCs) were then obtained by stimulating adherent cells with 800-1000IU/ml GM-CSF and 400-600IU/ml IL-4 in AIM-V medium (purchased from GIBCO) for 6 days. On day 6, 200ng/ml IL6, 10ng/ml TNF-. alpha.10 ng/ml IL-1. beta. and 1. mu.g/ml PGE2 were added. On day 7, mature dendritic cells were harvested. Phenotypic identification was performed by flow cytometry and the DC cells were identified to meet the requirements of subsequent experiments.
2.2 preparation of DC extracellular exosomes
Use of AIM-V Medium at 5% CO 2 Culturing the above DC cells in an incubator at 37 deg.C, and when the cell confluence reaches 90%, replacing the fresh medium containing 10% FBS at 5% CO 2 Culturing in 37 deg.C incubator for 48 hr; collecting the culture medium, and centrifuging at 4 deg.C for 5min, 10min and 20min at 500g, 3000g and 5000g respectively to remove free living cells, dead cells and cell debris; filtering the obtained solution by using a 0.22 mu m filter membrane to remove large-particle impurities; then, the mixture was centrifuged at 10000g at 4 ℃ for 60min, the supernatant was discarded, the mixture was washed 3 times with sterile PBS, and the mixture was centrifuged at 4 ℃ for 90min at 10000g, and the precipitate was collected to obtain dendritic cell Dex particles. And (3) as shown in figure 1, the Dex particles are concentrated in diameter between 80 and 150nm and uniform and stable in particle size by using scanning electron microscope analysis detection. The Dex particles were stored in a-80 ℃ freezer for a long period of time to meet the needs of subsequent experiments.
2.3 Induction expression of DC cells and preparation of post-induction Dex
In earlier studies, it was found that it is difficult to obtain satisfactory anti-tumor effect by directly using Dex secreted from natural DC cells, which may be caused by insufficient anti-tumor substances contained in Dex due to ineffective activation of natural DC cells, thus affecting anti-tumor effect in vitro experiments. Therefore, different inducers are adopted to activate DC cells and promote the secretion of antitumor active substances in the invention.
Carcinoembryonic antigen (CEA) and Carbohydrate Antigen (CA)125 are reported to be important tumor markers of breast cancer, and are closely related to the occurrence and development of breast cancer, so the above substances are used as DC cell induction reagents in this embodiment; it has also been reported that cytokines such as IL-12, IL-10, IFN-. gamma.and TGF-. beta.can also participate in the activation process of immune cells and induce immune cellsDifferentiation and maturation, and secretion of exosome are stimulated, so in the embodiment, the breast cancer marker and the cytokine are combined to form an induction reagent, and Dex secretion is stimulated to improve the anti-tumor effect. CEA + CA125, CEA + CA125+ IL-12, CEA + CA125+ IL-10, CEA + CA125+ IFN-gamma, CEA + CA125+ TGF-beta are used as inducing reagents respectively, after the DC cell fusion degree reaches 90%, a fresh culture medium containing 10% FBS is replaced, the inducing reagents are added simultaneously, the concentration of each inducing reagent is 20ng/mL of CEA, 20ng/mL of CA125 12520 ng and 10ng/mL of cytokine respectively, the CEA and the CA125 are purchased from abcam company, and the IL-12, the IL-10, the IFN-gamma and the TGF-beta are purchased from near-shore protein company. At 5% CO 2 After incubation for 48h at 37 ℃ in an incubator, Dex was extracted (as described in section 2.2).
Example 3DC cell exosomes in vitro anti-tumor experiments
In order to study the inhibition effect of Dex on breast cancer provided by the present invention, the breast cancer MCF-7 cell line was used as the experimental subject in this example.
3.1 tumor killing ability
MCF-7 cells were recovered (preserved by the present inventors), and DMEM medium containing 10% FBS was added thereto at 37 ℃ with 5% CO 2 Culturing under saturated humidity, and repeating 2-3 generations to revive cells. Cells were harvested, cell density adjusted using fresh medium, seeded into 96-well cell culture plates at 1X 10 per well 5 Individual cell, 37 ℃ and 5% CO 2 After incubation at saturation humidity for 12h, equal volumes of DMEM medium (control group) and Dex (CEA + CA125) produced by DC cells induced by native Dex, CEA and CA125, Dex (CEA + CA125+ IL-12) produced by DC cells induced by CEA, CA125 and IL-12, Dex (CEA + CA125+ IL-12) produced by DC cells induced by CEA, CA125 and IL-10, Dex (CEA + CA125+ IFN-gamma) produced by DC cells induced by CEA, CA125 and IFN-gamma, Dex (CEA + CA125+ IFN-beta) produced by DC cells induced by TGF-beta, were added to the cells at a final concentration of 20 ng/mL. After addition of inducer, 5% CO at 37 ℃ 2 Incubate 48h at saturated humidity. Then, 20. mu.L/well of 2.5mg/mL MTT solution was added to each well, incubated for 4h in the dark, the cell culture solution was discarded, 150. mu.L of dimethyl sulfoxide was added to each well, shaken in a shaker for 15min, and the absorbance value was read at 490 nm. Tumor cell survival rate was calculated (experimental group)Absorbance/control absorbance) x 100%.
As shown in fig. 2, although natural Dex has a certain tumor-inhibiting effect, the effect is weak and it is difficult to achieve a satisfactory effect; after CEA and CA125 are used for induction treatment, the anti-tumor activity of DC cells is stimulated, and the generated Dex also has stronger tumor killing capacity, so that the survival rate of breast cancer cells is reduced; but after IL-12 or IFN-gamma is added into an inducer, the induced Dex tumor inhibition capacity is further enhanced, wherein CEA + CA125+ IL-12 shows the strongest activation capacity; IL-10 appears not to produce significant promoting or inhibiting effect, TGF-beta appears to play a certain role in tumor killing inhibition, and the tumor killing ability of Dex appears to be inhibited in the CEA + CA125+ TGF-beta inducer combination, which is related to the fact that TGF-beta can play different roles in different stages of the tumor development process, and TGF-beta can promote tumor growth and development in some cases. The experiment shows that CEA + CA125+ IL-12 is an effective inducing reagent combination, and Dex formation is induced in the subsequent experiment in the way.
Example 4 Dex-antibody Complex preparation
To further enhance the anti-tumor effect, in this example, a Dex-nano anti HER2 complex was prepared. According to the invention, phospholipid polyethylene glycol succinimidyl ester (DSPE-PEG-NHS) is selected as a cross-linking agent to connect Dex and a targeted HER2 antibody, and the cross-linking agent is a biocompatible cross-linking agent, so that the antibody and Dex can be effectively connected to form a binary compound, the biological activities of the antibody and Dex can be retained to the greatest extent, the damage to a biomolecule and a phospholipid bilayer with a micro-vesicular structure is prevented, and the anti-tumor effect is favorably exerted.
The conventional crosslinking reaction conditions are reaction at low temperature (such as 4 ℃), which can prepare Dex-antibody complex, but has longer reaction time and lower product yield, and is not beneficial to large-scale production, and the invention uses the preparation method in journal literature (Targeted Exosomes for Drug Delivery: Biomanufecting, Surface labeling, and differentiation, Yingnan Si et al, Biotechnol J.2020Jan; 15(1): e1900163.) to use medium-high temperature (37 ℃) in the reaction stage of Dex and DSPE-PEG-NHS-nano anti 2 complex, which is closer to cell survival environment, can maintain stability of microvesicle, greatly shorten reaction time, and improve preparation efficiency and product yield.
The method comprises the following specific steps: uniformly mixing phospholipid polyethylene glycol succinimidyl ester dissolved in organic solvent dimethyl sulfoxide and HER2 nano antibody according to a ratio of 10:1, stirring and mixing for 30min in a sterile environment at 4 ℃, then reacting at 4 ℃ overnight, centrifuging at 5000rpm for 15min to remove unreacted phospholipid polyethylene glycol succinimidyl ester (DSPE-PEG-NHS) to obtain a phospholipid polyethylene glycol succinimidyl ester-HER 2 nano antibody compound (DSPE-PEG-NHS-nano anti HER 2); dissolving the dendritic cell exosome in sterile phosphate buffer solution to obtain dendritic cell exosome solution, uniformly mixing the compound and the dendritic cell exosome according to the ratio of 5:1, gently sucking and uniformly mixing at 4 ℃, reacting for 4h at 37 ℃, and centrifuging at 4500rpm for 5min to obtain the dendritic cell exosome carrying the targeted HER2 nano antibody.
By adopting the method, a compound Dex-nano anti HER2 obtained by combining natural Dex with an antibody and a compound inDex-nano anti HER2 obtained by combining Dex generated by inducing CEA + CA125+ IL-12 with the antibody are respectively prepared for the follow-up verification of the anti-tumor effect.
Example 5 tumor killing experiment of Dex-antibody Complex
5.1 in vitro tumor killing experiment
The killing effect of the Dex-antibody complex on breast cancer MCF-7 cells is detected, and the specific method refers to section 3.1. As shown in fig. 3, after the Dex and the antibody form a compound, the anti-tumor capability is greatly enhanced, the tumor survival rate is reduced to about 40%, and compared with the single Dex tumor killing capability, the tumor killing capability is improved by about one time; the anti-tumor activity of the Dex antibody compound induced and activated by CEA + CA125+ IL-12 is further improved, which shows that the mode can effectively improve the anti-tumor capability.
5.2 Regulation of Fas protein expression
FAS (also known as Apo-1 or CD95), a potent member of the death receptor family, plays a key role in apoptotic signaling in many cell types, interacts with its FAS ligand (FASL) to trigger the death signaling cascade, subsequently inducing apoptotic cell death. Evidence suggests that many tumors exhibit down-regulation or loss of function of FAS, resulting in resistance to immune system-induced death signals, and increased expression of FASL-mediated immune privileges, and thus, decreased FAS expression and/or increased FASL expression may contribute to malignant transformation and progression. In this section, the change condition of Fas gene after exosome treatment is detected by Western blot method, and the specific steps are as follows:
extracting total protein of the breast cancer cells after treatment (the treatment method is the same as the treatment method in section 3.2) by using a protein extraction kit (purchased from BestBio Biotech company); performing protein gel electrophoresis under the conditions of gel concentration 2030min, voltage 90V, gel separation 1h and voltage 110V; soaking the PVDF membrane in methanol for activation for 5min, and then rotating the membrane under 25V voltage by a semi-dry transfer method to transfer the protein from the gel to the PVDF membrane; incubating the membrane with the front side up for 2h in a blocking solution at 37 ℃ with shaking, and then washing 3 times with TBST; the PVDF membrane was immersed in a primary antibody dilution (antibody purchased from Abcam) with the front side up, incubated overnight at 4 ℃ and washed 3 times with TBST; the PVDF membrane is soaked in a secondary antibody diluent (the antibody is purchased from Abcam company) in a right-side-up manner, incubated for 2h at 37 ℃ and washed 3 times by TBST; and (5) dripping a color developing agent, and carrying out photographing detection.
As shown in figure 4, after the Dex antibody compound provided by the invention is used for treatment, the Fas protein expression amount in cells is remarkably improved, and particularly in an inDex-nano anti HER2 treatment group, Fas is expressed at a high level, so that the Fas protein can effectively mediate the tumor cell apoptosis process and play a strong tumor killing role.
5.3 in vivo antitumor Effect
Breast cancer MCF-7 cells are cultured and expanded, as described in section 3.1. When the cell fusion degree reaches more than 90%, collecting cells and adjusting the cell concentration to 5 × 10 6 One cell per mL, 100 mu L of cell suspension is inoculated to the subcutaneous part of the axilla of the anterior left limb of a Balb/c nude mouse by a syringe until the tumor volume grows to 100-200mm 3 And then used in subsequent experiments.
The tumor nude mice are randomly divided into three groups, 20 mice in each group are respectively injected with 100 mug/kg Dex-nano anti HER2, 100 mug/kg inDex-nano anti HER2 and physiological saline with the same volume, the administration is carried out once every 2 days for 4 weeks, the tumor volume is measured every week, and cervical dislocation and sacrifice are carried out after the experiment is finished. As shown in figure 5, the Dex antibody compound provided by the invention can effectively inhibit tumor growth, and the anti-tumor effect of the inDex-nano anti HER2 is better than that of the Dex-nano anti HER2 from the second week, the trend is more obvious compared with in vitro experiments, probably because DC cells induced and activated by CEA + CA125+ IL-12 can be more adaptable to tumor microenvironment and play an anti-tumor role, and the Dex serving as a drug delivery vector can effectively carry an anti-HER 2 antibody so as to play a targeted anti-tumor role.
While this invention has been particularly shown and described with references to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
Sequence listing
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Claims (9)

1. A pharmaceutical composition for treating malignant tumor, characterized in that the pharmaceutical composition comprises dendritic cell exosomes carrying HER 2-targeting antibodies, the exosomes are connected with the HER 2-targeting antibodies through phospholipid polyethylene glycol succinimidyl ester cross-linking agents, the HER 2-targeting antibodies are nanobodies, and the nanobodies comprise CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3.
2. The pharmaceutical composition of claim 1, wherein the nanobody has an amino acid sequence shown in SEQ ID NO. 4.
3. The pharmaceutical composition of claim 1, wherein the step of preparing said dendritic cell exosomes comprises: use of AIM-V Medium at 5% CO 2 Culturing human DC cells in 37 deg.C incubator, changing fresh culture medium when cell fusion degree reaches 90%, and culturing in 5% CO 2 Culturing in 37 deg.C incubator for 48 hr; collecting the culture medium, and removing free living cells, dead cells and cell debris in the culture medium by adopting a gradient centrifugation method; filtering the obtained solution by using a 0.22 mu m filter membrane to remove large-particle impurities; then performing ultracentrifugation at 10000g at 4 ℃ for 60min, discarding the supernatant, washing with sterile PBS for 3 times, performing ultracentrifugation at 10000g at 4 ℃ for 90min, and collecting the precipitate to obtain the dendritic cell exosome particles.
4. The pharmaceutical composition of claim 3, wherein the step of preparing said dendritic cell exosomes comprises: adding inducer during DC cell culture process, wherein the inducer comprises one or more of carcinoembryonic antigen CEA, carbohydrate antigen CA125, IL-12, IL-10, IFN-gamma and TGF-beta.
5. The pharmaceutical composition of claim 4, wherein the inducing agent comprises carcinoembryonic antigen CEA, carbohydrate antigen CA125, and IL-12.
6. The pharmaceutical composition of claim 1, wherein the step of linking the dendritic cell exosomes to a HER 2-targeting antibody comprises: uniformly mixing phospholipid polyethylene glycol succinimidyl ester dissolved in organic solvent dimethyl sulfoxide and HER2 nano antibody according to the proportion of 1:10-10:1, stirring and mixing for 30min in a sterile environment at 4 ℃, then reacting overnight at 4 ℃, centrifuging for 10-15min at 5000rpm to remove unreacted phospholipid polyethylene glycol succinimidyl ester, and obtaining the phospholipid polyethylene glycol succinimidyl ester-HER 2 nano antibody compound; dissolving the dendritic cell exosome in sterile phosphate buffer solution to obtain dendritic cell exosome solution, uniformly mixing the compound and the dendritic cell exosome according to the proportion of 1:5-5:1, gently sucking and uniformly mixing at 4 ℃, reacting for 2-4h at 37 ℃, and centrifuging at 4500rpm for 5min to obtain the dendritic cell exosome carrying the targeted HER2 nano antibody.
7. Use of the pharmaceutical composition of any one of claims 1 to 6 for the preparation of an anti-tumor medicament.
8. The use according to claim 7, wherein the tumor is selected from one or more of breast cancer, lung cancer, stomach cancer, colorectal cancer, prostate cancer, head and neck cancer, pancreatic cancer, lymphoma, leukemia and/or myeloma.
9. The use according to claim 8, wherein the tumor is selected from breast cancer.
CN202210642703.9A 2022-06-08 2022-06-08 Pharmaceutical composition containing dendritic cell exosomes and application thereof Pending CN114848838A (en)

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CN116751745A (en) * 2023-08-09 2023-09-15 北京圣美细胞生命科学工程研究院有限公司 Combined immune cell exosome polypeptide regeneration factor and application thereof

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CN113403276A (en) * 2021-06-23 2021-09-17 河北大学 Antibody functionalized exosome preparation and preparation method and application thereof

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CN113403276A (en) * 2021-06-23 2021-09-17 河北大学 Antibody functionalized exosome preparation and preparation method and application thereof

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* Cited by examiner, † Cited by third party
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CN116751745A (en) * 2023-08-09 2023-09-15 北京圣美细胞生命科学工程研究院有限公司 Combined immune cell exosome polypeptide regeneration factor and application thereof

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