CN114410686A

CN114410686A - Engineered exosome in-situ nano vaccine and preparation method and application thereof

Info

Publication number: CN114410686A
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: 2022-04-29
Anticipated expiration: 2041-12-24
Also published as: CN114410686B

Abstract

The invention discloses a recombinant vector for over-expressing alpha-LA, a cell line for over-expressing alpha-LA, exosomes, a nano vaccine prepared by utilizing engineered and modified breast cancer source exosomes enriched with alpha-lactalbumin, 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, releases tumor-associated antigens, Hiltonol and damage-associated molecular patterns (DAMPs), and activates 1 type conventional dendritic cells in situ, thereby triggering strong tumor-specific CD8⁺T cell response, show effective antitumor activity. The nano vaccine provides a new method for killing tumor cells in a targeted manner, provides a new mode for in-situ activation of the 1-type conventional dendritic cells, and provides a new idea for improving the immunotherapy of breast cancer.

Description

Engineered exosome in-situ nano vaccine and preparation method and application thereof

Technical Field

The invention belongs to the technical field of tumor immunotherapy drugs, and particularly relates to an engineered exosome in-situ nano vaccine as well as a preparation method and application thereof.

Background

Dendritic Cells (DCs), the most potent antigen presenting cells, are the core of the initiation and regulation of innate and adaptive immunity in the tumor microenvironment. Therefore, various vaccines targeting DCs have been developed to improve cancer immunotherapy and performed various clinical trials, but are still difficult to popularize clinically. At present, a DCs vaccine widely researched is a DCs nano vaccine consisting of an antigen and an adjuvant, and the vaccine directly activates and mobilizes natural DCs subgroups at multiple sites in vivo and is an important method for enhancing the anti-tumor effect of DCs. However, due to the lack of targeting of the adjuvant, the adjuvant is easy to induce the nonspecific activation of the systemic immune system, and the local or systemic inflammatory reaction is triggered, so that potential immunotoxic side effects which are difficult to avoid exist. Furthermore, another major challenge of this strategy is to identify the optimal immunodominant tumor antigen to elicit an effective tumor-specific immune response. Solving the problems can help the popularization and the application of the DCs vaccine in clinic.

Immunogenic Cell Death (ICD) enhances tumor antigen exposure and promotes release of immunostimulatory contents within tumor cells, and whole tumor cell ICD lysates have been investigated for development of autologous DC vaccines. The induction of tumor cells ICD in vivo would provide a broad antigen repertoire for DCs vaccines, avoiding the identification of immunodominant antigens. The cDC1s can effectively process and cross-present tumor-associated exogenous antigen, and further activate CD8⁺T cells and respond, however, DCs are often immature in the tumor microenvironment, resulting in less efficient presentation of tumor antigens, and therefore, there is a need to deliver immune-adjuvanted TLR3 agonists that effectively activate cDC1s in vivo into the tumor microenvironment, 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 to activate tumor-infiltrating cDC1s in situIn an ideal manner.

In recent years, exosome is a natural drug delivery carrier due to its characteristics of low cytotoxicity, capability of maximally improving drug bioavailability and easiness in bioengineering, and drugs developed by using exosome have been subjected to a plurality of clinical tests, thereby further proving its clinical transformation value. Thus, tumor-derived exosomes (Texs) can serve as cell-free therapeutic vectors, co-delivering ICD inducers and TLR3 agonists into the tumor microenvironment to activate cDC1s in situ.

Triple Negative Breast Cancer (TNBC) is a specific breast cancer subtype that does not express Estrogen Receptor (ER), Progesterone Receptor (PR) and human epidermal growth factor receptor 2(HER2), accounting for approximately 15% of all breast cancers. Due to the special molecular phenotype, TNBC is insensitive to hormone therapy or molecular targeted therapy, and the curative effect of conventional postoperative adjuvant radiotherapy and chemotherapy is poor. Median survival time after TNBC transfer is 13.3 months, and postoperative recurrence rate is up to 25%, and development of a new TNBC treatment strategy is urgently needed. There is a strong correlation between the infiltration of the cDC1s and good prognosis of TNBC, therefore, the above-mentioned therapeutic strategy of activating cDC1s in situ is expected to improve the therapeutic predicament of TNBC. In order to improve the targeting of the tumor exosomes to TNBC, the exosomes need to be engineered. Alpha-lactalbumin (alpha-LA) is a mammary gland-specific immunodominant antigen expressed in most human breast cancers, and the enrichment of alpha-LA on the surface of exosomes helps to enhance exosome targeting ability and immunogenicity. In view of the above, the invention designs the alpha-LA engineered exosome in-situ nano vaccine carrying the ICD inducer ELANE and the TLR3 agonist Hiltonol, and the alpha-LA engineered exosome in-situ nano vaccine is used for immunotherapy for treating and improving breast cancer.

Disclosure of Invention

The purpose of the invention is as follows: based on the technical problem, the invention provides an engineered exosome in-situ nano vaccine and a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme that the method comprises the following aspects:

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

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

The breast cancer cells include, but are not limited to, triple negative breast cancer cells MDA-MB-231 cells, and other breast cancer cells are also included, so long as the breast cancer cells can express the above-mentioned alpha-lactalbumin or secrete the above-mentioned alpha-lactalbumin exosomes, and the invention is in the protection scope.

As another aspect of the present invention, there is provided an exosome extracted from the cell line according to

claim

2 or 3.

As another aspect of the present invention, the present invention provides an engineered exosome in-situ nano-vaccine comprising the exosome.

As another aspect of the invention, the invention provides a preparation method of the engineered exosome in-situ nano-vaccine, which comprises the following steps:

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

2) extracting the exosome enriched with the alpha-lactalbumin from the cell culture supernatant;

3) and (3) electrotransfering an immunogenic cell death inducer ELANE and a TLR3 agonist Hiltonol into the obtained exosome by an electroporator to obtain the engineered exosome in-situ nano vaccine.

It is to be noted that the invention uses genetic engineering means to transform exosomes to accurately deliver ELANE and Hiltonol to tumor cells, and induces strong CD8 by triggering immunogenic cell death of the tumor cells, activating type 1 conventional dendritic cells in situ⁺T cell reaction, effectively inhibiting tumor growth.

As another aspect of the invention, the invention provides an application of the recombinant vector for over-expressing alpha-LA, the cell line for over-expressing alpha-LA, the exosome or the engineered exosome in-situ nano-vaccine in preparing a medicament for treating breast cancer or breast cancer immunotherapy.

As another aspect of the invention, the invention provides a medicament for treating breast cancer, which comprises the exosome or the vaccine.

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

Experiments further show that the engineered exosome in-situ nano vaccine can effectively inhibit tumor growth in an immunocompetent mouse and in a tumor patient-derived organoid, the in-vitro and in-vivo immune activation effect of the engineered exosome in-situ nano vaccine is stronger than that of a free immune adjuvant Hiltonol, the engineered exosome in-situ nano vaccine does not cause a systemic inflammatory reaction similar to that of the free immune adjuvant Hiltonol in vivo, and the mechanism of the tumor inhibition effect comprises but is not limited to the targeting induction of the engineered exosome in-situ nano vaccine on tumor cells to cause immunogenic cell death, further, the mechanism of the tumor inhibition effect comprises but is not limited to the in-situ activation of type 1 conventional dendritic cells by the engineered exosome in-situ nano vaccine, so that tumor-specific CD8 is activated⁺T cells, further inducing strong tumor killing.

Has the advantages that: compared with the prior art, the invention has the following advantages: the invention successfully enriches alpha-lactalbumin on the surface of the exosome derived from the breast cancer cells by a genetic engineering means, so that the modified exosome specifically targets the breast cancer cells. Using this engineered breast cancer-derived exosome, we efficiently delivered ELANE and Hiltonol to tumor cells, induced intense CD8 by triggering immunogenic cell death of the tumor cells, activating conventional dendritic cells of type 1 in situ, inducing⁺T cell response, therebyEffectively inhibit the growth of tumor.

Drawings

FIG. 1 identification of MDA-MB-231 cell lines stably overexpressing alpha-LA;

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

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

FIG. 4, detection of the in vitro immune activation ability of the nano-vaccine HELA-Exos;

FIG. 5, in vivo evaluation of the anticancer effect of the nano-vaccine HELA-Exos;

FIG. 6, in vivo safety evaluation of the Nanoprotein HELA-Exos vaccine;

FIG. 7 evaluation of the in vivo immune activation ability of the Nanoprotein HELA-Exos vaccine;

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

Detailed Description

The following examples are intended to illustrate the invention in further detail, but are not intended to limit the invention. The experimental methods of the present invention are all referred to the experimental conditions proposed in "molecular biology laboratory Manual of precise edition" (edited by F.M. Osber et al, published by scientific Press 2005).

The following are specific media components used in the examples:

1. t cell culture medium:

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

2. Human breast cancer organoid culture medium

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

The preparation method of the alpha-LA engineered exosome in-situ nano vaccine wrapping ELANE and Hiltonol comprises the following steps:

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

The human alpha-LA full-length coding sequence (Access: CR542017) was cloned from total RNA of MDA-MB-231 cells (this laboratory deposit) and into the lentiviral expression vector pCDH-CMV-puro (JHepatol.2017 Oct; 67(4): 739-. Human 293T cells were seeded in 6cm dishes and cultured for 24h, and then human 293T cells were co-transfected with pCDH-CMV-puro-alpha-LA, pMD2.G (J Gene Med.2018Jul; 20(7-8): e3027.) (Collection in this laboratory) and psPAX2(JGene Med.2018Jul; 20(7-8): e3027.) (Collection in this laboratory) plasmids at a ratio of 2:1:1 by DNA transfection reagent. The virus was harvested 48 hours later and titrated for subsequent MDA-MB-231 cell transfection. MDA-MB-231 cells were seeded in 12-well plates and transfected with α -LA expressing lentivirus, repeated every 18 hours for 1, 3 total transfections, and finally α -LA overexpressing MDA-MB-231 cells were screened with puromycin (2 μ g/mL). The MDA-MB-231 cell line (alpha-LA/MDA-MB-231) stably over-expressing alpha-LA was identified by Western blotting, and the results are shown in FIG. 1, wherein alpha-LA/MDA-MB-231 is highly expressed in comparison with the control cell.

2. Exosome (LA-Exos) extraction surface enriched with alpha-LA

Adherent MDA-MB-231 cells stably overexpressing alpha-LA were washed with PBS, the culture medium was changed to a culture medium containing 1% FBS, and culture supernatants were collected after 48 hours. The collected cell supernatants were centrifuged at 4 ℃ at 300g for 10 minutes to remove cells remaining in the culture, and at 3,000g for 30 minutes to remove cell debris remaining in the culture. An overspeed rotor was selected and centrifuged at 100,000g for 70min at 4 ℃. The supernatant was removed and resuspended in precooled PBS and then ultracentrifuged again at 100000g at 4 ℃ for 70 min. The precipitate is the exosome (LA-Exos) with alpha-LA enriched on the surface, and the obtained exosome can be stored for a long time at the temperature of minus 80 ℃.

3. Preparation of nano vaccine HELA-Exos

LA-Exos, human neutrophil elastase (ELANE) (Abcam, ab280938) and Hiltonol (Oncovir Inc.) are mixed in 300 μ l of electroporation buffer solution, electroporation is carried out in a 4mm dish according to electroporation parameters of 400V and 150 μ F, after electroporation, the exosome is centrifuged by PBS at 4 ℃ and 100,000g for 70min for 2 times, so as to obtain nano vaccine HELA-Exos, and the obtained HELA-Exos is characterized. As shown in FIG. 2, the nano-vaccine HELA-Exos was structurally complete under a transmission electron microscope, the particle size distribution was measured by a nano-particle sizer, the HELA-Exos mean particle size was increased to about 113nm compared to LA-Exos, the nano-flow results showed that HELA-Exos successfully encapsulated ELANE and Hiltonol, and the Western blot results showed that HELA-Exos retained the exosome marker components and was significantly enriched for α -LA compared to the untransfected MDA-MB-231 cell-derived exosomes (Texs) (obtained by the ultracentrifugation method described above from the culture supernatant of untransfected MDA-MB-231 cells).

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

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

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

Cell uptake efficiency was assessed by flow cytometry and fluorescence confocal experiments using DiI-labeled HELA-Exos or Texs incubated with a cell mixture of MDA-MB-231 cells and human Peripheral Blood Mononuclear Cells (PBMCs) for 2 hours, or DiI-labeled HELA-Exos or Texs added after MDA-MB-231 cells and human lung cancer cells a549 cells were plated, respectively, as shown in fig. 3A-B, HELA-Exos significantly improved targeting ability compared to Texs, and MDA-MB-231 cells taken up more nano-vaccine HELA-Exos than PBMC and a549 cells, indicating that HELA-Exos has excellent targeting specificity for breast cancer cells.

2. Nano vaccine HELA-Exos in-vitro cell targeted killing and ICD induction function evaluation

And (3) incubating Texs and HELA-Exos with breast cancer cells MDA-MB-231, MCF7, MDA-MB-435, SKBR-3 and normal breast epithelial cells MCF10A for 48 hours respectively, and quantifying the cell killing efficiency of the HELA-Exos by calcein-AM living cell fluorescent staining, wherein the result is shown in fig. 3C-D, after the HELA-Exos treatment, only a small amount of stained living cells remain in the breast cancer cells, the fluorescent quantitative killing efficiency is detected by using a microplate reader, the killing efficiency of the HELA-Exos to the breast cancer cells reaches over 80 percent, and the HELA-Exos hardly kills the normal breast epithelial cells. The capacity of HELA-Exos to induce ICD in breast cancer cells is evaluated by detecting the cell membrane translocation of killed MDA-MB-231 cells of Calreticulin (CRT) and the release of ATP and HMGB1 in cell culture supernatant, and the result is shown in figures 3D-F, and the HELA-Exos remarkably increases the cell membrane translocation of MDA-MB-231 cells and the release of ATP and HMGB1, and effectively induces the MDA-MB-231 cells to generate ICD.

3. Evaluation of in vitro immune activation capability of nano vaccine HELA-Exos

MDA-MB-231 cells were co-cultured with PBMC-derived DCs and pretreated 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. After 48 hours, the cells were harvested and analyzed for cellular immune activation by flow cytometry, and the results are shown in FIG. 4, where CD141 (a marker for cDC1 s) and HLA-A2(MHC class I molecules) in DCs were increased in fluorescence intensity in the HELA-Exos treated group, while CD1c (a marker for cDC2 s) and HLA-DR (MHC class II molecules) showed no significant difference. In the HELA-Exos-treated group, CD8⁺Increased production of granzyme B and perforin in T cells, increased expression of CD69 (activation marker), and CD8⁺Expression of PD1 was reduced in T cells. The above results indicate that HELA-Exos induces ICD in breast cancer cells, thereby promoting maturation of cDC1s and further activating CD8⁺T cells, causing their activation to a killer phenotype.

Example 3: evaluation of in vivo anticancer effect and immune activation capability of nano vaccine HELA-Exos

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

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

The MDA-MB-231 cell line stably expressed by luciferase is used for in situ inoculation of immune active Balb/c mice, the tumor grows for about 21 days, and the volume reaches about 100mm³Treatment with DMEM basal medium, Hiltonol or HELA-Exos was started, tumor volume was monitored, mice were anesthetized 30 days after treatment for in vivo imaging, and mice were subsequently sacrificed. The results are shown in FIG. 5, in vivo tumor imaging and tumor growth curve results FIGS. 5A-C show significant inhibition of HELA-Exos treatmentTumor growth in mice. In addition, the expression of a proliferation marker Ki67 is reduced, the expression of an apoptosis marker Caspase-3 and the expression of an ICD marker ICD are increased in tumor tissues of mice treated by the HELA-Exos, and the nano vaccine HELA-Exos is shown to effectively inhibit the tumor growth in immunocompetent orthotopic tumor mice.

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

After the heart, liver, spleen, lung and kidney of the treated mouse are sliced, H & E staining is carried out, the result is shown in figure 6A, organic lesions do not occur in the important organs of the mouse after HELA-Exos treatment, the liver and kidney toxicity indexes ALT, AST, BUN and CREA levels in the serum of the mouse are detected, the result is shown in figure 6B, and the liver and kidney function damage of the mouse is not caused after HELA-Exos treatment. The serum levels of the inflammation indicators IL-6, IL-12 and TNF-alpha were measured in mice, and the results are shown in FIG. 6B, where the HELA-Exos treated mice reduced systemic inflammatory responses induced by Hiltonol treatment.

3. Evaluation of immune activation capability of nano vaccine HELA-Exos in mice

The immune microenvironment of the tumor tissues of the treated mice was analyzed by tissue immunofluorescence staining, flow cytometry and immunohistochemical staining, and the results are shown in fig. 7. Tissue immunofluorescent staining and flow cytometry results FIGS. 7A-B show that HELA-Exos treatment significantly increased the tissue microenvironment and subset of cDC1 in draining lymph nodes in mice (CD11 c)⁺CD103⁺) And (4) infiltrating. 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 production of granzyme B and perforin in T cells, increased expression of CD69, and CD8⁺Expression of PD1 was reduced in T cells. These results indicate that HELA-Exos can effectively promote infiltration and in situ maturation of the cDC1 subpopulation and enhance antitumor CD8 within the microenvironment⁺T cell immunization.

Example 4: evaluation of immune activation capability and anti-cancer effect of nano-vaccine HELA-Exos in patient-derived organoids

The immune activation capability and the anti-cancer effect evaluation of the nano vaccine HELA-Exos in the organoid of the patient source comprise the following four parts:

1. tissue-derived organoid culture for breast cancer patients

Cutting the breast cancer tissue obtained by surgical resection into small pieces, preparing tumor tissue single cell suspension by a tumor separation kit, and removing residual red blood cells by using red blood cell lysate for cracking. The cell pellet was resuspended in 10mg/mL matrigel and the process was run on ice. Subsequently, 40. mu.L of cell suspension was punched out of the plate, the droplets were solidified in an incubator at 37 ℃ for 20 minutes, and finally 400. mu.L of human breast cancer organoid medium was added thereto at 37 ℃ with 5% CO₂The culture is carried out in a humidifying incubator, and the culture medium of the human breast cancer organoid is replaced every 4 days.

2. Establishing PBMCs and autologous tumor organoid co-culture system of breast cancer patient

Dissociating the tissue-derived organoid of the breast cancer patient into single cells, resuspending the single cells in a T cell culture medium, adding autologous PBMCs, co-culturing the cells in the T cell culture medium containing IL-2, adding a DMEM basal medium, Hiltonol or HELA-Exos for co-stimulation, changing the solution 2 to 3 times per week, supplementing the IL-2, Hiltonol or HELA-Exo, re-stimulating the PBMCs with a fresh tissue-derived organoid of the breast cancer patient every week, and co-culturing the tissue-derived organoid for 2 weeks.

3. Evaluation of immune activation capability of nano vaccine HELA-Exos in co-culture system

After two weeks of co-culture, the flow cytometry and immunofluorescence staining techniques were used to assess the immune activation of the nano-vaccine HELA-Exos in the co-culture system, and the results are shown in FIGS. 8A-D, where the organoids in the co-culture system showed CD141 in the HELA-Exos treated group⁺cDC1s and CD8⁺Increased T cell infiltration and CD8⁺A significant increase in granzyme B and perforin production in T cells indicates that HELA-Exos can effectively enhance cDC1s activation and effectively initiate subsequent tumor-specific CD8⁺T cell responses.

4. Evaluation of tumor killing capability 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 tumor growth inhibition by HELA-Exos was evaluated by staining organoids with viable and dead cells, as shown in fig. 8E-F, MTS assay results showed significant reduction in organoid cell viability in the HELA-Exos treated group, and staining with viable and dead cells showed significant increase in dead cells inside organoids in the HELA-Exos treated group. The results show that the nano vaccine HELA-Exos obviously inhibits the growth of autologous tumor organoids, effectively induces cell death, and shows effective anti-tumor activity in breast cancer.

Claims

1. A recombinant vector for overexpressing alpha-LA, wherein the recombinant vector is obtained by cloning an alpha-LA gene into a lentiviral expression vector.

2. A cell line overexpressing α -LA, wherein the cell line overexpressing α -LA is obtained by transfecting a breast cancer cell with the lentiviral expression vector of claim 1.

3. The cell line overexpressing α -LA according to claim 2, wherein said breast cancer cells are triple negative breast cancer cells MDA-MB-231 cells.

4. An exosome, wherein the exosome is extracted from the cell line of claim 2 or 3.

5. An engineered exosome in situ nano-vaccine, comprising an exosome according to claim 4.

6. The method for preparing the engineered exosome in-situ nano-vaccine according to claim 5, characterized by comprising the following steps:

1) transfecting a breast cancer cell with the recombinant vector for over-expressing alpha-LA as claimed in claim 1 to prepare a stable cell line for expressing alpha-lactalbumin;

7. Use of the recombinant vector for overexpressing α -LA according to claim 1, the cell line for overexpressing α -LA according to any one of claims 2 to 3, the exosome according to claim 4 or the engineered exosome in situ nano-vaccine according to claim 5 for the preparation of a medicament for the treatment of breast cancer or breast cancer immunotherapy.

8. A medicament for the treatment of breast cancer, comprising an exosome according to claim 4 or a vaccine according to claim 5.

9. The medicament for treating breast cancer according to claim 8, wherein the breast cancer is triple negative breast cancer.