WO2023109657A1

WO2023109657A1 - Exosome for promoting tumor infiltration of t lymphocyte and preparation method therefor

Info

Publication number: WO2023109657A1
Application number: PCT/CN2022/137699
Authority: WO
Inventors: 李韵清; 陈有海; 孔艺
Original assignee: 深圳先进技术研究院; 中国科学院深圳理工大学(筹)
Priority date: 2021-12-15
Filing date: 2022-12-08
Publication date: 2023-06-22
Also published as: CN114349845B; CN114349845A

Abstract

Provided are an exosome for promoting tumor infiltration of a T lymphocyte and a preparation method therefor. By modifying the membrane structure of a T lymphocyte exosome, the binding of a T lymphocyte and a tumor cell can be promoted, and the exosome uptake of a tumor is also facilitated. The membrane of the exosome contains proteins expressed by target genes CXCR2, CXCR3, and CD47, and thus, the membrane of an improved exosome contains rich chemokine receptors CXCR2 and CXCR3, the infiltration rate of the T lymphocyte and a tumor tissue can be promoted, and the stability of the exosome is improved. Further provided is an efficient purification method.

Description

An exosome that promotes tumor infiltration of T lymphocytes and preparation method thereof

technical field

The invention belongs to the technical field of tumor treatment, and in particular relates to an exosome that promotes the tumor infiltration of T lymphocytes and a preparation method thereof.

Background technique

Tumor immunotherapy has received great attention recently, and was rated as the top ten scientific and technological breakthroughs of the year by Science in 2013, and has broad application prospects. Despite promising results in the treatment of hematological malignancies and malignant melanoma, immunotherapy has had little effect in most solid tumors.

Low tumor infiltration of immune cells is one of the major bottlenecks for anti-tumor cell therapy. As an important natural media particle between cells, exosomes participate in activities such as cell migration and chemotaxis. T lymphocytes are the final effector cells of anti-tumor immunity. Adoptive cell therapy (ACT), which reinfuses T lymphocytes activated by tumor-associated antigen (TAA) to treat tumors, has been widely used in recent years. focus on. However, the tumor tissue infiltration of T lymphocytes is low, which greatly reduces the effect of immunotherapy. How to increase the infiltration degree of T lymphocytes in tumor tissue is an urgent problem to be solved.

Exosomes are membranous vesicles with a diameter of approximately 30–160 nm released by cells. Exosomes were once thought of as debris and metabolic waste produced by cells. However, recent studies have shown that exosomes can carry cell-specific information and transmit the corresponding information to other cells and distant tissues. As the "vehicle" of cell-cell communication, exosomes play an important regulatory role in both physiological and pathological states. In addition to lipids and proteins, there are also genetic materials such as mRNA and miRNA in exosomes. As a nano-scale "carrier", the application value of exosomes in anti-tumor drug/immunity/gene therapy vectors has also received great attention in recent years. As nano-scale vesicles secreted by cells, exosomes have the ability to efficiently deliver proteins and nucleic acids. As a carrier for drug delivery, exosomes have the following advantages: ① The phospholipid bimolecular membrane structure of exosomes has good stability in vivo and can effectively Protect the drug it carries; ②Exosomes from self-derived exosomes cause a low rate of immune rejection after injection into the body; ③Exosomes have certain targeting properties and can fuse with target cell membranes to release drugs into cells.

technical problem

However, the research on the application of exosomes in drug delivery has just started, and the most significant problem is the low rate of uptake of exosomes in tumor tissues. Most of the drug-loaded exosomes entering clinical trials increase the tumor uptake rate of exosomes through high injection volume, but this method does not fundamentally solve the problem of low tumor uptake rate of exosomes. High exosome injections are very likely to stimulate the body's innate immunity. It has been proved by experiments that in a clinical trial of weekly administration of 1.3x10 ¹³ dendritic cell exosomes in the treatment of non-small cell lung cancer, one-third of patients developed NK Cell lysis and MAGE-specific T lymphocytes were significantly elevated and indicated the risk of triggering a cytokine storm.

technical solution

In view of the defects of related technologies, the present invention proposes an exosome that promotes the tumor infiltration of T lymphocytes and a preparation method thereof. By modifying the membrane structure of T lymphocyte exosomes, the modified T lymphocytes secrete The body can not only recruit circulating T lymphocytes to infiltrate tumor tissue, but also has the potential clinical value of improving the tumor microenvironment. The invention can accurately induce circulating T lymphocytes to enter tumor tissue, solve the common problem of low infiltration degree of T lymphocytes in cancer treatment, and improve the efficacy of immune cell therapy.

The first aspect of the present invention provides an exosome that has the ability to promote the tumor infiltration of T lymphocytes, and the membrane of the exosome contains a protein expressed by the target gene CXCR2.

Further, the exosome membrane also contains the protein expressed by the target gene CXCR3.

Further, the exosome membrane also contains the protein expressed by the target gene CD47.

The second aspect of the present invention provides a recombinant vector, which is a eukaryotic expression vector or a viral vector, and one or more of target genes CXCR2, CXCR3 or CD47 are recombined on the vector.

Further, the vector is a recombinant virus vector.

Further, the recombinant viral vector is a recombinant lentiviral vector.

The third aspect of the present invention provides a recombinant cell, comprising the above-mentioned recombinant vector.

Further, the recombinant cells are recombinant T lymphocytes.

Further, the recombinant T lymphocytes are Jurkat cells.

The fourth aspect of the present invention provides a method for preparing the above-mentioned exosomes, comprising the following steps:

S1. Recombining the target genes CXCR2, CXCR3 and CD47 into the lentiviral expression vector, transfecting Jurkat cells, using 5 μg/mL ampicillin penicillin to screen the cell lines highly expressing GFP, and performing subculture and proliferation for purification;

S2. Add 10 ng/mL IL-2 to stimulate Jurkat at 37°C and 5% CO ₂ for 8 h, then centrifuge at 1500g x 5 min, and take the culture supernatant, which is the culture medium containing Exo-C;

S3. Use a PALL 20mL ultrafiltration tube with a molecular weight cut-off=4k, centrifuge for 25 k x 20 min; use the exosome extraction kit, and extract exosomes;

S4. Through Avanti The homogenizer is used for particle screening, and Exo-C with a pore size <200 nm is screened out particle size.

Beneficial effect

The improved exosomes of the present invention have multiple beneficial effects: it can promote the tumor infiltration of drug-loaded exosomes, improve the targeting and efficiency of treatment, and increase the uptake rate of tumor cells. It has been shown that abundant chemokine receptors are expressed on exosome membranes, which can induce T lymphocytes to migrate against the direction of exosome concentration gradient. The improved exosome membrane contains abundant chemokine receptors CXCR2 and CXCR3, which can induce T lymphocytes to migrate against the direction of exosome concentration gradient, and promote the movement of T lymphocytes to tumor tissues, thereby increasing the infiltration rate. Using an efficient purification method, the purity of exosome extraction is as high as 97% and the yield is increased by more than 20 times, surpassing the traditional ultracentrifugation and single exosome kits. The first exosome carrier containing chemokines targeting CXCL7 and CXCL10 was prepared, which greatly improved the tumor infiltration of T lymphocyte exosomes. High expression of CD47 greatly reduces the possibility of macrophages phagocytizing exosomes and increases the stability of exosomes. By modifying the membrane structure of exosomes of T lymphocytes, the present invention enhances the tumor targeting of exosomes containing drugs and enhances the ability of exosomes to move to tumors, and can continuously and steadily promote the growth of immune T lymphocytes. Tumor infiltration. That is, T lymphocyte exosomes containing drugs can induce circulating T lymphocytes to enter tumor tissue and kill cells at the same time. T lymphocytes reach the highest value, and unnecessary side effects such as peripheral tissue inflammation are minimized.

Description of drawings

Figure 1A is a schematic diagram of the process of constructing recombinant cells;

Figure 1B is a schematic diagram of the construction of a lentivirus containing a target gene;

Figure 2A is a schematic diagram of an improved exosome extraction and purification scheme;

Figure 2B is a schematic diagram of the purity of exosomes extracted without the improved protocol;

Figure 2C is a schematic diagram of the purity of exosomes extracted through the protocol improvement;

Figure 3A is a schematic diagram of the experimental construction of the Transwell test of exosomes;

Figure 3B is a schematic diagram of the chemotaxis promoting effect of Jurkat exosomes as the representative research object;

Figure 3C is a schematic diagram of the effect of serum concentration on the chemotaxis of exosomes;

Figure 3D is a schematic diagram of the comparison of exosome-promoting chemotaxis rate.

Embodiments of the present invention

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not constitute a conflict with each other.

The present invention enhances the tumor targeting of exosomes containing drugs and enhances the ability of exosomes to move to tumors by modifying the structure on the exosome membrane of T lymphocytes, and can continuously and smoothly promote the tumor infiltration of immune cells . That is, T lymphocyte exosomes containing drugs can not only accurately induce circulating T lymphocytes to enter tumor tissue, but also increase the infiltration of T lymphocytes in tumor tissue and improve the targeting of exosomes. In addition, the invention can be used as adjuvant therapy for low-response population of anti-tumor therapy.

The present invention prepares an exosome that can efficiently bind to tumor cells, and the membrane of the exosome contains proteins expressed by target genes CXCR2 and CXCR3. In addition to the proteins expressed by the target genes CXCR2 and CXCR3, the exosome membrane also contains the protein expressed by the target gene CD47.

The present invention prepares a T lymphocyte, the exosome membrane of the T lymphocyte is modified, and the membrane contains proteins expressed by CXCR2 and CXCR3.

The invention relates to a recombinant vector, which contains target genes CXCR2 and CXCR3. The structure of the recombinant lentiviral vector carrying the target gene is shown in Figure 1B.

The preparation and purification methods of exosomes and the confirmation of chemotaxis experiments adopt the methods mentioned below.

The preparation method of exosomes is as follows:

Expression of exosomes: The target genes CXCR2, CXCR3 and CD47 were recombined into the lentiviral expression vector, and then transfected into Jurkat T lymphocytes (Jurkat cells for short). After 48 hours, the cell lines highly expressing GFP were screened with 5 μg/mL ampicillin (Fig. 1A and Fig. 1B), then subcultured, proliferated, and purified. The heparan sulfate was electrotransferred onto the Exo-C membrane by electroporation.

Exosome production: Add 10 ng/mL IL-2 to stimulate Jurkat at 37°C and 5% CO ₂ for 8 h, centrifuge at 1500g x 5 min, and take the supernatant of the culture medium, that is, the culture containing Exo-C base. Concentration and purification of Exo-C: use a PALL 20mL ultrafiltration tube with a molecular weight cut-off = 4k, and centrifuge at 25 kx for 20 min. Exosomes were extracted using an exosome extraction kit (Thermal Fisher) according to the manufacturer's instructions.

Secondary purification: Exo-C particles with a pore size of <200 nm were screened by an Avanti homogenizer.

The exosome membrane prepared by the above method contains proteins expressed by the target genes CXCR2 and CXCR3, and CXCR2 is highly expressed and binds to the ligand CXCL7 of CXCR2. CXCR3 is highly expressed and binds to CXCR3 ligand CXCL10. The levels of CXCL7 and CXCL10 in the blood of patients with various solid tumors are significantly higher. It can promote the combination of drug-containing exosomes and tumor cells.

The exosomes prepared by the above method have more beneficial effects: they can promote the combination of drug-containing exosomes and tumor cells, improve the targeting and efficiency of treatment, and increase the uptake rate of tumor cells. It has been shown that abundant chemokine receptors are expressed on exosome membranes, which can induce T lymphocytes to migrate against the direction of exosome concentration gradient. The improved exosome membranes contain abundant chemokine receptors. CXCR2 and CXCR3 can induce T lymphocytes to migrate against the direction of exosome concentration gradient, and promote the movement of T lymphocytes to tumor tissues, thereby increasing the infiltration rate. Using an efficient purification method, the purity of exosome extraction is as high as 97% and the yield is increased by more than 20 times, surpassing the traditional ultracentrifugation and single exosome kits. The first exosome carrier targeting CXCL7 and CXCL10 was prepared, which greatly improved the binding of exosomes to tumor cells. High expression of CD47 greatly reduces the possibility of macrophages phagocytizing exosomes and increases the stability of exosomes.

Chemotactic effect confirmed:

Experimental scheme-comparison of exosome extraction and purification schemes, as shown in Figure 2A, 2B, and 2C.

The purity of exosomes treated without the improved purification protocol was 74%. Use a 30 k PEG and 2.5 k cut-off weight dialysis membrane for dialysis, and dialyze and concentrate the medium used for culturing cells, such as 45 mL of RPMI medium concentrated to 6 mL. The exosome extraction kit Thermofisher was used for extraction, and the protein content was measured to be 1.52 mg/mL *400 uL (about 0.6 mg purified product), centrifuged at 10 k, 21 min + 19.4 k, 5 min. The obtained exosome products conform to the particle size range of exosomes ~100%, as shown in Figure 2C, the peak shape is clear. Exosomes were extracted from untransfected healthy human primary T lymphocytes, CAR-T, and Jurkat cells by Invitrogen kits. Extraction method: 5 x ¹⁰⁵ cells were cultured in a transparent six-well plate. After culturing for 24 hours under standard conditions, Jurkat cells were stimulated by adding 100 U/mL IL-2, and after 24 hours, exocrine was extracted according to the instructions of the kit. body. A Malvern particle size analyzer test was performed to confirm the particle size.

Experimental protocol - Transwell, as shown in Figure 3A.

Upper chamber: 1.0 x 10 ⁵ healthy Jurkat cells were pipetted, diluted in 200 μL of serum-free RPMI medium, and seeded in the upper chamber of a 24-well 5 um PC membrane-transwell chamber. The movement of human T lymphocytes and Jurkat cells was observed under a confocal microscope. The chemotactic effect of exosomes on Jurkat cells was tested by determining how many Jurkat cells migrated to the high-concentration lower chamber area.

Experimental conclusion: Jurkat exosomes (19%), that is, the experimental group showed significantly higher T-lymphocyte chemotaxis than the blank control group (0%) in the Transwell experiment (p=0.03), and exosomes were not added in the case of exosomes In the lower chamber, the number of Jurkat cells measured in the lower chamber was 0, as shown in Figure 3B. Therefore, in the absence of exosomes, Jurkat cells were restricted from migrating in a short period of time in the medium stimulated by serum and exosomes. In the experiment of adding exosomes, Jurkat exosomes showed a chemotaxis ability that was absolutely higher than that of the empty control group, and it was not due to random movement caused by serum starvation, as shown in Figure 3C. The phenomenon tested in Transwell shows that the chemotactic effect of exosomes is not caused by the release of substances in the membrane by exosomes. The serum-free medium supernatant of exosomes co-cultured for 4 hours was extracted and filtered with an ultrafiltration tube (cutoff value =3k, PALL) filtered out without exosomes, extracted and added to the lower chamber of Transwell, it was not found that it could promote the migration of Jurkat to the lower chamber (Figure 3D).

It is easy for those skilled in the art to understand that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, All should be included within the protection scope of the present invention.

Claims

An exosome that promotes tumor infiltration of T lymphocytes is characterized in that the membrane of the exosome contains a protein expressed by the target gene CXCR2.
The exosome according to claim 1, wherein the membrane of the exosome also contains a protein expressed by the target gene CXCR3.
The exosome according to claim 1 or 2, wherein the membrane of the exosome also contains a protein expressed by the target gene CD47.
A recombinant vector, characterized in that the vector is a eukaryotic expression vector or a virus vector, and one or more of target genes CXCR2, CXCR3 or CD47 are recombined on the vector.
The recombinant vector according to claim 4, wherein the vector is a recombinant viral vector.
The recombinant vector according to claim 5, wherein the recombinant viral vector is a recombinant lentiviral vector.
A recombinant cell, characterized in that it comprises the recombinant vector according to claim 4 or 5 or 6.
The recombinant cell according to claim 7, wherein the recombinant cell is a recombinant T lymphocyte.
The recombinant cell according to claim 8, wherein the recombinant T lymphocytes are Jurkat cells.
A method for preparing exosomes according to any one of claims 1-3, comprising the steps of:

S1. Recombining the target genes CXCR2, CXCR3 and CD47 into the lentiviral expression vector, transfecting Jurkat cells, using 5 μg/mL ampicillin penicillin to screen the cell lines highly expressing GFP, and performing subculture and proliferation for purification;

S2. Add 10 ng/mL IL-2 to stimulate Jurkat at 37°C and 5% CO 2 for 8 h, then centrifuge at 1500g x 5 min, and take the culture supernatant, which is the culture medium containing Exo-C;

S3. Use a PALL 20mL ultrafiltration tube with a molecular weight cut-off=4k, centrifuge for 25 k x 20 min; use the exosome extraction kit, and extract exosomes;

S4. Through Avanti The homogenizer is used for particle screening, and Exo-C with a pore size <200 nm is screened out particle size.