CN114410686B

CN114410686B - Engineering exosome in-situ nanometer vaccine and preparation method and application thereof

Info

Publication number: CN114410686B
Application number: CN202111599982.7A
Authority: CN
Inventors: 汪付兵; 黄兰祥; 袁纯辉; 荣媛
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2023-10-03
Anticipated expiration: 2041-12-24
Also published as: CN114410686A

Abstract

The invention discloses a recombinant vector for over-expressing alpha-LA, a cell line and exosome for over-expressing alpha-LA, a nano vaccine prepared by using breast cancer source exosome enriched with alpha-lactalbumin after engineering transformation, and a preparation method and application thereof. The nano vaccine of the invention specifically targets tumor tissues through alpha-lactalbumin, induces tumor cells to generate immunogenic cell death and releases tumor related antigens, hiltonol and injury related molecular patterns (DAMPs), activates type 1 conventional dendritic cells in situ, and further induces strong tumor specific CD8 ⁺ T cell response, exhibiting potent anti-tumor activity. The nanometer vaccine provides a new method for targeting killer tumor cells, a new mode for activating type 1 conventional dendritic cells in situ, and a new thought for improving the immunotherapy of breast cancer.

Description

Engineering exosome in-situ nanometer vaccine and preparation method and application thereof

Technical Field

The invention belongs to the technical field of tumor immunotherapy medicaments, and in particular relates to an engineering exosome in-situ nanometer vaccine, a preparation method and application thereof.

Background

Dendritic Cells (DCs), the most potent antigen presenting cells, are the core for the initiation and regulation of innate and adaptive immunity in the tumor microenvironment. Thus, a variety of vaccines targeting DCs have been developed to improve cancer immunotherapy and have been subjected to a number of clinical trials, but are still difficult to popularize clinically. The DCs vaccine which is widely studied at present is a DCs nano vaccine composed of antigen and adjuvant, and the vaccine directly activates and mobilizes natural DCs subgroup at a plurality of sites in the body, thus being an important method for enhancing the anti-tumor effect of DCs. However, because of the lack of targeting of adjuvants, nonspecific activation of the systemic immune system is easily induced, local or systemic inflammatory responses are elicited, and there are potentially immunotoxic side effects that are unavoidable. In addition, another major challenge of this strategy is the recognition of optimal immunodominant tumor antigens to elicit an effective tumor-specific immune response. Solving the problems is helpful for the popularization and application of DCs vaccine in clinic.

Immunogenic Cell Death (ICD) enhances tumor antigen exposure and promotes release of immunostimulatory content within tumor cells, and studies have been made to use whole tumor cell ICD lysates for development of autologous DC vaccines. Inducing tumor cell ICDs in vivo would provide a broad antigen pool for DCs vaccines, while avoiding the identification of immunodominant antigens. cDC1s is effective in treating and cross-presenting tumor-associated exogenous antigens, thereby activating CD8 ⁺ T cells and responses, however, DCs are often immature in the tumor microenvironment, resulting in less efficient presentation of tumor antigens, and thus, there is a need to deliver immune adjuvant TLR3 agonists to the tumor microenvironment that are effective in activating cDC1s in vivo, thereby ameliorating CD8 in the tumor microenvironment ⁺ Recruitment, infiltration, and function of T cells. Thus, targeted delivery of ICD inducers and TLR3 agonists to tumor tissue would be an ideal way to activate tumor infiltration dcs 1s in situ.

In recent years, exosomes are natural drug delivery vehicles due to the characteristics of low cytotoxicity, capability of improving the bioavailability of drugs to the greatest extent and easiness in bioengineering, and drugs developed by using exosomes have a plurality of clinical trials, so that the clinical transformation value of the exosomes is further proved. Thus, tumor-derived exosomes (Texs) can be used as cell-free therapeutic vectors to co-deliver ICD inducers and TLR3 agonists into the tumor microenvironment to activate dcs 1s in situ.

Triple Negative Breast Cancer (TNBC) is a specific subtype of breast cancer that does not express Estrogen Receptor (ER), progestogen Receptor (PR) and human epidermal growth factor receptor 2 (HER 2), accounting for about 15% of all breast cancers. Because of the special molecular phenotype, TNBC is insensitive to hormone treatment or molecular targeting treatment, and the conventional postoperative auxiliary radiotherapy and chemotherapy has poor curative effect. Median survival time after TNBC transfer is 13.3 months, postoperative recurrence rate is as high as 25%, and development of new TNBC treatment strategies is urgently needed. There is a strong correlation between the infiltration of dcs 1s and the good prognosis of TNBC, therefore, the above therapeutic strategy for in situ activation of dcs 1s is expected to improve the treatment dilemma of TNBC. In order to improve the targeting of the tumor exosomes to TNBC, it is necessary to engineer exosomes. Alpha-lactalbumin (alpha-LA) is a mammary gland-specific immunodominant antigen expressed in most human breast cancers, and enrichment of alpha-LA on the surface of the exosomes helps to enhance exosome targeting ability and immunogenicity. In view of this, the present invention devised an alpha-LA engineered exosome in situ nanovaccine loaded with ICD inducer ELANE and TLR3 agonist hiltenonol for use in the treatment of improved immunotherapy of breast cancer.

Disclosure of Invention

The invention aims to: based on the above, the technical problem to be solved by the invention is to provide an engineering exosome in-situ nanometer vaccine, and a preparation method and application thereof.

In order to achieve the above purpose, the following technical scheme is adopted in the invention:

as a first aspect of the present invention, the present invention provides a recombinant vector overexpressing α -LA, which is obtained by cloning an α -LA gene into a lentiviral expression vector.

As another aspect of the invention, the invention provides an α -LA overexpressing cell line obtained by transfecting breast cancer cells with the lentiviral expression vector.

Wherein the breast cancer cells include, but are not limited to, triple negative breast cancer cells MDA-MB-231, and other breast cancer cells are also included, as long as the breast cancer cells can express the alpha-lactalbumin or secrete the alpha-lactalbumin exosome.

As another aspect of the present invention, the present invention provides an exosome extracted from the cell line of claim 2 or 3.

As another aspect of the invention, the invention provides an engineered exosome in situ nanovaccine comprising said exosome.

As another aspect of the present invention, the present invention provides a method for preparing the engineered exosome in situ nano vaccine, comprising the steps of:

1) Transfecting the recombinant vector over-expressing the alpha-LA into breast cancer cells to prepare a stable cell line expressing the alpha-lactalbumin;

2) Extracting from the cell culture supernatant to obtain an exosome enriched in alpha-lactalbumin;

3) Electrotransformation of the immunogenic cell death inducer ELANE and the TLR3 agonist hiltenol into the obtained exosomes by means of an electroporation instrument, an engineered exosome in situ nanovaccine was obtained.

It should be noted that, the present invention uses genetic engineering to modify exosomes to precisely deliver ELANE and Hiltonol to tumor cells, and induces apoptosis of tumor cell immunogenic cells, activates type 1 conventional dendritic cells in situ, and induces strong CD8 ⁺ T cell reaction, effectively inhibit tumor growth.

As another aspect of the invention, the invention provides the use of said recombinant vector overexpressing α -LA, said cell line overexpressing α -LA, said exosome or said engineered exosome in situ nanovaccine in the preparation of a medicament for the treatment of breast cancer or immunotherapy of breast cancer.

As another aspect of the present invention, there is provided a medicament for treating breast cancer, said medicament comprising an exosome as described or a vaccine as described.

Wherein the breast cancer includes, but is not limited to, triple negative breast cancer, it should be noted that the medicament of the present invention can also treat other breast cancers, as long as the breast cancer cells can express the above alpha-lactalbumin or can secrete the above alpha-lactalbumin exosomes.

The invention is further described by experimentsThe in-situ nano-vaccine of the engineering exosome effectively inhibits the growth of tumors in immunocompetent mice and organs derived from tumor patients, the in-situ nano-vaccine of the engineering exosome has stronger immune activation effect in vitro and in vivo than that of the free immune adjuvant Hiltonol, and the in-situ nano-vaccine of the engineering exosome does not cause systemic inflammatory reaction similar to that of the free immune adjuvant Hiltonol in vivo, the mechanism of the tumor inhibition effect of the invention comprises but is not limited to the targeting induction of the immune cell death of tumor cells by the nano-vaccine of the engineering exosome in-situ, and the mechanism of the tumor inhibition effect of the invention comprises but is not limited to the in-situ activation of the conventional dendritic cells of the type 1 by the nano-vaccine of the engineering exosome in-situ, so as to activate the tumor specific CD8 ⁺ T cells, further induce strong tumor killing.

The beneficial effects are that: compared with the prior art, the invention has the following advantages: the invention successfully enriches alpha-lactalbumin on the surface of the exosome from breast cancer cells by a genetic engineering means, so that the modified exosome specifically targets the breast cancer cells. With this engineered breast cancer-derived exosome, we effectively delivered ELANE and hiltenol to tumor cells, inducing strong CD8 by eliciting tumor cell immunogenic cell death, activating conventional dendritic cells in situ ⁺ T cell response, thereby effectively inhibiting tumor growth.

Drawings

FIG. 1, MDA-MB-231 cell line identification of stable over-expression of α -LA;

FIG. 2, analysis and identification of nano-vaccine HELA-Exos;

FIG. 3, targeting analysis of nano vaccine HELA-Exos and in vitro cell killing specificity identification;

FIG. 4, nanometer vaccine HELA-Exos in vitro immune activation ability assay;

FIG. 5, in vivo anticancer efficacy assay of the nanovaccine HELA-Exos;

FIG. 6, in vivo safety assessment of nanovaccine HELA-Exos;

FIG. 7, in vivo immune activation ability assessment of the nanovaccine HELA-Exos;

FIG. 8, evaluation of immune activating ability and anticancer effect of nano vaccine HELA-Exos in patient-derived organoids.

Detailed Description

The following examples are provided to illustrate the invention in further detail, but are not intended to limit the invention. The experimental methods of the invention are all based on the experimental conditions suggested by the "Fine programming molecular biology laboratory Manual" (published by scientific Press in 2005, the main editions of F.M. Osbur et al).

The following are the specific media components used in the examples:

1. t cell medium:

composition of the components	Concentration of	Goods number	Suppliers (suppliers)
				Ultraglutamine I	2mM	BE17-605E/U1	Lonza
Penicillin/Streptomycin	100μg/mL	15140-122	Invitrogen
				male human AB serum	10％	1810-0001	SeraCare
RPMI 1640 basal medium	1×	SH30027.FS	HyClone

2. Human breast cancer organoid culture medium

Example l: preparation of alpha LA engineered exosome in situ nano vaccine wrapping ELANE and Hiltonol

A method for preparing an alpha-LA engineered exosome in situ nanovaccine encapsulating ELANE and hiltenol, the method comprising the steps of:

1. construction of MDA-MB-231 cell lines stably overexpressing alpha-LA

The full length coding sequence for human alpha-LA was cloned from the total RNA of MDA-MB-231 cells (deposited in this laboratory) (Accession: CR 542017) and cloned into the lentiviral expression vector pCDH-CMV-puro (Jhepatol. 2017Oct;67 (4): 739-748.) (deposited in this laboratory). Human 293T cells were inoculated in 6cm dishes and cultured for 24h, then human 293T cells were co-transfected with pCDH-CMV-puro-alpha-LA, pMD2.G (J Gene Med.2018Jul;20 (7-8): e 3027.) (this laboratory deposit) and psPAX2 (JGene Med.2018Jul;20 (7-8): e 3027.) (this laboratory deposit) plasmids in a 2:1:1 ratio by DNA transfection reagent. Viruses were harvested 48 hours later and titrated for subsequent MDA-MB-231 cell transfection. MDA-MB-231 cells were seeded on 12-well plates and transfected with α -LA expressing lentiviruses 1 repeat every 18 hours, 3 total transfections, and finally the α -LA overexpressing MDA-MB-231 cells were screened with puromycin (2. Mu.g/mL). The MDA-MB-231 cell line (alpha-LA/MDA-MB-231) stably overexpressing alpha-LA was identified by western blotting, and the results were shown in FIG. 1, in which alpha-LA/MDA-MB-231 was highly expressed as compared to control cells.

2. Surface-enriched alpha-LA exosome (LA-Exos) extraction

The adherent MDA-MB-231 cells which had stably expressed α -LA were washed with PBS, the culture medium was replaced with a medium containing 1% FBS, and the culture supernatant was collected after 48 hours. The collected cell supernatant was centrifuged at 300g for 10 minutes at 4℃to remove the cells remaining in the culture broth, and at 3,000g for 30 minutes to remove the cell debris remaining in the culture broth. An overspeed rotor was selected and centrifuged at 100,000g for 70min at 4 ℃. The supernatant was removed, resuspended in pre-chilled PBS and centrifuged at 100000g for 70min at 4℃again. Precipitating to obtain exosome (LA-Exos) with surface enriched alpha-LA, and storing the exosome at-80deg.C for a long period.

3. Preparation of nano vaccine HELA-Exos

LA-Exos, human neutrophil elastase (ELANE) (Abcam, ab 280938) and Hiltonol (Oncovir Inc.) were mixed in 300. Mu.l electroporation buffer, electroporation was performed in 4mm dishes with electroporation parameters of 400V and 150. Mu.F, after electroporation, the exosomes were washed 2 times with PBS at 4℃and 100,000g centrifugation for 70min, thus obtaining the nanovaccine HELA-Exos, and the resulting HELA-Exos was characterized. The results are shown in FIG. 2, the nano vaccine HELA-Exos has complete structure under a transmission electron microscope, the average particle size of HELA-Exos is increased to about 113nm compared with that of LA-Exos, the nano flow type result shows that HELA-Exos successfully wraps ELANE and Hiltonol, the Western blotting result shows that HELA-Exos retains an exosome marker component and is enriched with alpha-LA in a large amount compared with exosomes (Texs) derived from untransfected MDA-MB-231 cells (obtained by the ultracentrifugation method in the culture supernatant of untransfected MDA-MB-231 cells).

Example 2: in vitro biological function evaluation of nano vaccine HELA-Exos

In vitro biological function evaluation of the nano vaccine HELA-Exos comprises the following three parts:

1. in vitro cell targeting ability evaluation of nano vaccine HELA-Exos

The efficiency of cell uptake was assessed by flow cytometry and fluorescence confocal experiments by either DiI-labelling HELA-Exos or Texs, incubating with a cell mixture of MDA-MB-231 cells and human Peripheral Blood Mononuclear Cells (PBMC) for 2 hours, or by adding DiI-labelling HELA-Exos or Texs after plating of MDA-MB-231 cells and human lung cancer cells A549 cells, respectively, and as a result, as shown in FIGS. 3A-B, HELA-Exos significantly improved targeting ability compared to Texs, and MDA-MB-231 cells uptake more nano-vaccine HELA-Exos than PBMC and A549 cells, indicating that HELA-Exos has excellent targeting specificity for breast cancer cells.

2. In vitro cell targeted killing and ICD induction function evaluation of nano vaccine HELA-Exos

The Texs and HELA-Exos are respectively incubated with breast cancer cells MDA-MB-231, MCF7, MDA-MB-435, SKBR-3 and normal breast epithelial cells MCF10A for 48 hours, and the cell killing efficiency of the HELA-Exos is quantified through calcein-AM living cell fluorescence staining, and the result is that after HELA-Exos treatment, only a small amount of stained living cells remain in the breast cancer cells, and the fluorescent quantitative killing efficiency is detected by using an enzyme-labeled instrument, wherein the killing efficiency of the HELA-Exos on the breast cancer cells reaches more than 80%, and the HELA-Exos hardly kills normal breast epithelial cells. The ability of HELA-Exos to induce breast cancer cells ICD was evaluated by detecting membrane translocation of Calreticulin (CRT) of the killed MDA-MB-231 cells and release of ATP and HMGB1 in the cell culture supernatant, and as a result, HELA-Exos significantly increased membrane translocation of MDA-MB-231 cells and release of ATP and HMGB1, as shown in FIG. 3D-F, to effectively induce ICD production of MDA-MB-231 cells.

3. Evaluation of in vitro immune activation ability of nanometer vaccine HELA-Exos

MDA-MB-231 cells were co-cultured with PBMC-derived DCs and pre-treated with DMEM basal medium, hiltonol or HELA-Exos for 24 hours, followed by addition of naive CD8 isolated from peripheral blood PBMC ⁺ T cells were co-cultured in 1640 medium supplemented with IL-2 for 48 hours. Cells were collected 48 hours later and analyzed for cellular immune activation using flow cytometry, as shown in FIG. 4, for CD141 (marker of cDC1 s) and in DCs in HELA-Exos treatment groupHLA-A2 (MHC-class I molecule) showed increased fluorescence intensity, whereas CD1c (marker of cDC2 s) and HLA-DR (MHC-class II molecule) showed no significant difference. CD8 in HELA-Exos treated group ⁺ Increased yields of granzyme B and perforin, increased expression of CD69 (activation marker), and CD8 in T cells ⁺ Expression of PD1 in T cells is reduced. The results indicate that HELA-Exos induces breast cancer cells to generate ICD, thereby promoting maturation of cDC1s and further activating CD8 ⁺ T cells, which cause their activation to a killing phenotype.

Example 3: evaluation of in vivo anticancer Effect and immune activation ability of nanometer vaccine HELA-Exos

The in vivo anticancer effect and immune activation capability evaluation of the nano vaccine HELA-Exos comprises the following three parts:

1. evaluation of tumor growth inhibition of nano vaccine HELA-Exos in immunocompetent in-situ tumor mice

Immunocompetent Balb/c mice were vaccinated in situ with luciferase stably expressed MDA-MB-231 cell line, and tumors grown for about 21 days to a volume of about 100mm ³ Tumor volumes were monitored by starting with DMEM basal medium, hilonol or HELA-Exos treatment, and anesthetized mice were subjected to live imaging 30 days after treatment, followed by sacrifice of the mice. Results in vivo tumor imaging and tumor growth curve results as shown in fig. 5A-C show that HELA-Exos treatment significantly inhibited tumor growth in mice. In addition, in tumor tissues of HELA-Exos treated mice, the expression of proliferation marker Ki67 is reduced, the expression of apoptosis marker Caspase-3 and ICD marker ICD is increased, and the nano vaccine HELA-Exos is shown to effectively inhibit tumor growth in immunocompetent in-situ tumor mice.

2. Safety evaluation of nano vaccine HELA-Exos in mice

The heart, liver, spleen, lung and kidney of the treated mice are sliced and then subjected to H & E staining, the result is shown in fig. 6A, the vital organs of the mice are not subjected to organic lesions after HELA-Exos treatment, the liver and kidney toxicity index ALT, AST, BUN and CREA level in the serum of the mice are detected, and the result is shown in fig. 6B, and the mice are not damaged in liver and kidney functions after HELA-Exos treatment. The results of detecting the levels of IL-6, IL-12 and TNF- α as indicators of inflammation in the serum of mice, as shown in FIG. 6B, reduced systemic inflammatory response caused by Hiltonol treatment in mice after HELA-Exos treatment.

3. Immune activation ability evaluation of nano vaccine HELA-Exos in mice

The tumor tissue immune microenvironment of the treated mice was analyzed by tissue immunofluorescence staining, flow cytometry and immunohistochemical staining, and the results are shown in fig. 7. FIG. 7A-B shows that HELA-Exos treatment significantly increased the micro-environment of mouse tissue and the cDC1 sub-population in draining lymph nodes (CD 11 c) ⁺ CD103 ⁺ ) Is impregnated with the solution. Flow cytometry and immunohistochemical staining results FIGS. 7C-E show that HELA-Exos treatment significantly increased CD8 in the mouse tissue microenvironment ⁺ T cell infiltration, and CD8 ⁺ Increased yields of granzyme B and perforin, increased expression of CD69, and CD8 in T cells ⁺ Expression of PD1 in T cells is reduced. These results indicate that HELA-Exos can effectively promote infiltration and in situ maturation of the cDC1 sub-population and enhance anti-tumor CD8 within the microenvironment ⁺ T cell immunity.

Example 4: immune activation ability and anticancer effect evaluation of nano vaccine HELA-Exos in patient-derived organoids

The immune activation capability and anticancer effect evaluation of the nano vaccine HELA-Exos in the patient-derived organoid comprises the following four parts:

1. tissue-derived organoid culture of breast cancer patients

The breast cancer tissue obtained by the surgical excision is cut into small blocks, a tumor tissue single cell suspension is prepared by a tumor separation kit, and residual red blood cells are removed by the lysis of red blood cell lysate. The cell pellet was resuspended in 10mg/mL matrigel and the process was run on ice. Subsequently, 40. Mu.L of the cell suspension was cast in a culture plate, the droplets were allowed to solidify in an incubator at 37℃for 20 minutes, and finally 400. Mu.L of human breast cancer organoid medium was added, at 37℃with 5% CO ₂ Is cultured in a humidified incubator, and the human breast cancer organoid medium is replaced every 4 days.

2. Establishing a PBMCs and autologous tumor organoids co-culture system for breast cancer patients

The tissue-derived organoids of the breast cancer patients are dissociated into single cells and resuspended in T cell medium, autologous PBMCs are added, co-cultured in T cell medium containing IL-2, and co-stimulated by adding DMEM basal medium, hiltonol or HELA-Exos, changing the fluid 2 to 3 times per week, including adding IL-2, hiltonol or HELA-Exo, and the PBMCs are re-stimulated with fresh tissue-derived organoids of the breast cancer patients for 2 weeks.

3. Immune activation ability evaluation of nano vaccine HELA-Exos in co-culture system

After two weeks of co-culture, the nano-vaccine HELA-Exos was evaluated for its ability to activate immunity in the co-culture system using flow cytometry and immunofluorescence staining techniques, and the results are shown in FIGS. 8A-D, in which organoids within the co-culture system showed CD141 in the HELA-Exos treated group ⁺ cDC1s and CD8 ⁺ Increased infiltration of T cells, and CD8 ⁺ The significant increase in granzyme B and perforin production in T cells suggests that HELA-Exos is effective in enhancing cDC1s activation and in initiating subsequent tumor-specific CD8 ⁺ T cell response.

4. Evaluation of tumor killing ability of nano vaccine HELA-Exos in co-culture system

After two weeks of co-culture, organoid cell viability was quantitatively determined by MTS cell proliferation and cytotoxicity kit (Biovision, K300), and organoids were subjected to live-dead cell staining to evaluate tumor growth inhibition by HELA-Exos, as shown in FIGS. 8E-F, and MTS assay results showed a significant decrease in organoid cell viability, while live-dead cell staining results showed a significant increase in organoid-internal dead cells in the HELA-Exos treated group. The result shows that the nano vaccine HELA-Exos remarkably inhibits the growth of autologous tumor organoids, effectively induces cell death and shows effective anti-tumor activity in breast cancer.

Claims

1. An engineered exosome in-situ nano-vaccine, characterized in that the preparation method of the engineered exosome in-situ nano-vaccine comprises the following steps:

1) Transfecting a recombinant vector which overexpresses alpha-LA into breast cancer cells to prepare a stable cell line expressing alpha-lactalbumin;

the recombinant vector for over-expressing the alpha-LA is obtained by cloning an alpha-LA gene into a lentiviral expression vector;

2. Use of the engineered exosome in situ nano-vaccine of claim 1 in the preparation of a medicament for treating breast cancer.

3. A medicament for the treatment of breast cancer, comprising the vaccine of claim 1.

4. A medicament for the treatment of breast cancer according to claim 3, wherein the breast cancer is triple negative breast cancer.