Construction of engineered erythrocyte extracellular vesicle drug delivery system and its application in leukemia

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the construction of an engineered erythrocyte extracellular vesicle drug delivery system and its application in leukemia. The present invention first provides a composite immunomodulator targeting leukemia cells, which is composed of a drug delivery system prepared from erythrocyte extracellular vesicles and contents; the drug delivery system includes erythrocyte extracellular vesicles and a targeting component connected thereto; the contents are small molecule drugs and small interfering RNA synergists. The composite immunomodulator has a composite effect of inducing immunogenic cell death and immune activation of leukemia cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the construction of an engineered erythrocyte extracellular vesicle drug-carrying system and its application in leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a highly heterogeneous malignant clonal disease. Treatments mainly include chemotherapy, targeted therapy, and hematopoietic stem cell transplantation. Currently, chemotherapy is still the first choice for most AML patients, especially young patients. If medium- and high-risk patients do not choose hematopoietic stem cell transplantation, the recurrence rate is as high as 60%-70%. Immunotherapy developed in recent years has become a powerful strategy for tumor treatment. Its characteristics are that it can activate the host immune response and is accompanied by the recognition and elimination of tumor cells. However, immunotherapy is currently developing rapidly in solid tumors, while the development of immunotherapy in AML is relatively lagging behind.

[0003] Currently, the main immunotherapy methods for AML include antibody-drug conjugates, immune checkpoint inhibitors, chimeric antigen receptor T cell (CAR-T) therapy and vaccines, but the overall treatment effect is not ideal. The main reason is that immunotherapy has more severe antigen presentation / recognition impairment and immunosuppressive microenvironment in AML. In addition, AML is also highly heterogeneous, and different patients respond differently to treatment at different stages of the disease, which is also the reason why the overall treatment effect of immunotherapy is not ideal.

[0004] The patent with publication number CN105412939A and invention name "A doxorubicin co-drug delivery system, its preparation method and application" discloses a co-drug delivery system for treating solid tumors. The co-drug delivery system carries active ingredients including doxorubicin and small interfering RNA. The co-drug delivery system is administered locally to targeted solid tumors, so that the small interfering RNA inhibits the expression of the Bmil gene in tumor cells. However, this drug delivery system is not suitable for AML because AML is difficult to treat by local administration. AML cells are mostly dispersed in peripheral blood, and traditional administration methods are easily retained in other normal organs, and the overall therapeutic effect is limited.

[0005] On the other hand, recent studies have shown that inducing immunogenic cell death (ICD) in tumor cells may be an effective strategy to improve tumor immunogenicity. Studies have shown that traditional local treatment methods (including local hyperthermia, radiotherapy or chemotherapy) can induce tumor immunogenic cell death. Among them, local area heat treatment is the most commonly used method to induce ICD. For example, 2D polypyrrole nanosheets are wrapped with carriers as NIR-II photothermal sensors. However, this local heat treatment method is more suitable for solid tumors.

[0006] In summary, it is necessary to propose new methods and strategies to improve the problems existing in the treatment of AML in existing technologies. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide an engineered erythrocyte extracellular vesicle drug delivery system and a construction method thereof and application in leukemia, so as to partially solve or alleviate the above-mentioned deficiencies in the prior art, and specifically adopt the following technical solutions.

[0008] A composite immunomodulator targeting leukemia cells, wherein the leukemia cells are leukemia cells with high expression of CLL1; the composite immunomodulator is composed of a drug delivery system prepared from erythrocyte extracellular vesicles and contents; the drug delivery system includes erythrocyte extracellular vesicles and a targeting component connected thereto, the targeting component is a polypeptide, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.1; the contents are small molecule drugs and small interfering RNA synergists; the small molecule drugs include doxorubicin, and the small interfering RNA is a small interfering RNA targeting IDO1.

[0009] Furthermore, the amino acid sequence of the small interfering RNA targeting IDO1 includes the sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

[0010] Furthermore, the dosage form of the composite immunomodulator is an injection.

[0011] Furthermore, the drug loading rate of the small molecule drug loaded in the drug delivery system is greater than 30%.

[0012] In some preferred embodiments, the drug loading rate of doxorubicin is 30%-33%.

[0013] Furthermore, the number of small interfering RNAs loaded in the drug delivery system is greater than 2000.

[0014] In some preferred embodiments, the number of the small interfering RNAs is 2000-2100.

[0015] Furthermore, the particle size of the composite immunomodulator is in the range of 150nm-300nm.

[0016] In some preferred embodiments, the particle size range is 170-175 nm.

[0017] Furthermore, the erythrocyte extracellular vesicles are derived from type O erythrocytes.

[0018] Furthermore, the composite immunomodulator has the combined effects of inducing immunogenic cell death and immune activation of leukemia cells.

[0019] The method for constructing the above-mentioned composite immunomodulator comprises the following steps:

[0020] S01: Extract and purify erythrocyte-derived extracellular vesicles;

[0021] S02: modifying the erythrocyte-derived extracellular vesicles, connecting the polypeptide fragment to DSPE-PEG2k-NHS via a -NH2 group to obtain PEP-PEG2k-DSPE, and then connecting PEP-PEG2k-DSPE to the erythrocyte-derived extracellular vesicles to obtain REVs-PEP;

[0022] S03: Loading small molecule drugs into the REVs-PEP by ultrasound, and then loading small interfering RNA targeting IDO1 by co-incubation.

[0023] Use of an engineered erythrocyte extracellular vesicle drug-carrying device in the preparation of a kit for evaluating the efficacy of leukemia immunomodulatory drugs, wherein the kit is used to evaluate the efficacy of leukemia immune drugs, wherein the efficacy includes inducing immunogenic cell death and / or immune activation of leukemia cells;

[0024] The engineered erythrocyte extracellular vesicle drug-carrying device comprises an erythrocyte extracellular vesicle and a targeting component connected thereto; the targeting component is a polypeptide, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.1;

[0025] The kit is provided with a detection reagent and a control reagent;

[0026] The detection reagent is an engineered erythrocyte extracellular vesicle drug-carrying device and a drug to be detected;

[0027] The control reagent is an engineered erythrocyte extracellular vesicle drug-carrying device and a control drug; the control drug is a small molecule drug and a small interfering RNA synergist; the small molecule drug is doxorubicin, and the small interfering RNA is a small interfering RNA targeting IDO1.

[0028] A summary of the sequences involved in the present invention.

[0029]

[0030] Beneficial technical effects

[0031] (1) The present invention provides a composite immunomodulator for CLL1-overexpressing leukemia, which can simultaneously induce immunogenic cell death and immune activation in leukemia cells, while also having high safety. Animal experiments have confirmed that after using the composite immunomodulator, experimental animals showed significantly lower leukemia burden and significantly prolonged lifespan.

[0032] (2) The present invention uses human type O red blood cells to prepare extracellular vesicles. Since the cells are widely available, the composite immunomodulator also has the potential for industrial production.

[0033] (3) The present invention screened for targeting components for leukemia cells with high CLL1 expression, thereby significantly enhancing the accuracy of targeting entry into the cells, thereby achieving precise drug delivery.

[0034] (4) The present invention not only proves that this composite immunomodulator has the function of inducing immunogenic cell death and immune activation of leukemia cells at the same time, but also confirms its therapeutic effectiveness in animal experiments. Therefore, this composite immunomodulator can be used as a reagent with proven effects to evaluate and control other drugs in the early stages of research and development. When the drug to be tested cannot achieve good immunomodulatory function in leukemia cells with high expression of CLL1, there is no need to conduct animal experiments, which can greatly save resources and costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0036] Figure 1A This is a representative TEM image of the red blood cell extracellular vesicles REVs isolated in one of the embodiments of the present invention (scale bar is 100 nm);

[0037] Figure 1B This is a particle size distribution diagram of the red blood cell extracellular vesicles REVs separated in one of the embodiments of the present invention;

[0038] Figure 1C The concentration standard curve and loading efficiency of Dox in one of the embodiments of the present invention (n = 3);

[0039] Figure 1D The concentration standard curve of Cy5-siIDO1 in one embodiment of the present invention;

[0040] Figure 1E is the number of siIDO1 molecules loaded on each REVs in one of the embodiments of the present invention (n = 3);

[0041] Figure 1FA representative TEM image and particle size distribution of REVs-Dox / siIDO1-PEP in one of the embodiments of the present invention (scale bar is 100 nm);

[0042] Figure 2 In one of the embodiments of the present invention, REVs-Dox / siIDO1-PEP is effectively absorbed by leukemia cells with high expression of CLL1 in vitro;

[0043] Figure 3 This is an evaluation of the cytotoxicity and gene transfection ability of REVs-Dox / siIDO1-PEP in one of the embodiments of the present invention;

[0044] Figure 4 This is a graph showing the experimental results of one of the embodiments of the present invention, that REVs-Dox / siIDO1-PEP is effectively absorbed by THP-1 cells in NCG mice;

[0045] Figure 5 This is a graph showing the experimental results of REVs-Dox / siIDO1-PEP being effectively absorbed by C1498 cells in C57BL / 6 in vivo in one of the embodiments of the present invention;

[0046] Figure 6 This is a diagram showing the experimental results of REVs-Dox / siIDO1-PEP inducing ICD in leukemia cells in one of the embodiments of the present invention;

[0047] Figure 7 This is a diagram showing the experimental results of REVs-Dox / siIDO1-PEP promoting in vivo immune activation in one of the embodiments of the present invention;

[0048] Figure 8 This is a diagram showing the in vivo anti-tumor effect of REVs-Dox / siIDO1-PEP in one of the embodiments of the present invention. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0050] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0051] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0052] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0053] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0054] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0055] Some drawings are detailed:

[0056] Figure 3In the figure, AB are the cytotoxicity of different concentrations of REVs (A) and REVs-Dox / siIDO1-PEP (B). C is the cell viability of THP-1, C1498 and AML primary cells after treatment with different concentrations of PBS / Dox / REVs-Dox / siIDO1-Pscr / REVs-Dox / siIDO1-PEP. D is the flow cytometry results showing the apoptosis rate of THP-1, C1498 and AML primary cells after treatment with PBS / Dox / REVs-Dox / siIDO1-Pscr / REVs-Dox / siIDO1-PEP. EG is the apoptosis rate of THP-1, C1498 and AML primary cells after treatment with REVs-Dox / siIDO1-Pscr and REVs-Dox / siIDO1-PEP. IDO1 The mRNA expression levels were expressed as mean ± SD (n = 3).

[0057] Figure 4 A is the flow cytometry result showing GFP expression in bone marrow, liver, peripheral blood and spleen + Uptake of DiD-labeled REVs by THP-1 cells (n = 5). B shows the uptake of DiD-labeled REVs by THP-1 cells in bone marrow, spleen, and liver. IDO1 mRNA expression levels (n = 3).

[0058] Figure 5 A is the flow cytometry result showing GFP expression in bone marrow, liver, peripheral blood and spleen + C1498 cells uptake DiD-labeled REVs (n = 5). B shows the uptake of DiD-labeled REVs by C1498 cells in bone marrow, spleen, and liver. IDO1 mRNA expression levels (n = 3).

[0059] Figure 6 In the figure, A is the immunofluorescence result showing the expression of CRT on the surface of THP-1 cells. BC is the ATP (B) level and HMGB1 (C) release of THP-1 cells after in vitro induction of ICD (n = 3). DE is the immunofluorescence result showing the expression of CRT (D) and HMGB1 on the surface of AML primary cells. F is the ATP level of AML primary cells after in vitro induction of ICD (n = 3). GH is the ATP (G) level and HMGB1 (H) release of C1498 cells after in vivo induction of ICD (n = 5). IJ is the representative flow cytometry (I) and quantitative analysis (J) results showing the maturation of DCs after co-culture with AML primary cells pretreated with PBS / Dox / REVs-Dox / siIDO1-PEP (CD80 + CD86 + ) (n = 3).

[0060] Figure 7 In the figure, A is the percentage of mature DC in the draining lymph nodes by flow cytometry. B is the percentage of CD3 + CD8 + T cell infiltration percentage. CD is the IFN-γ (C) and Ki67 (D) positive CD8 in the bone marrow of mice after administration. + EF is the percentage of CD8 T cells in the bone marrow of mice after administration. + Expression of PD1 (E) and TIM3 (F) on T cells.

[0061] Figure 8 In the figure, A is a schematic diagram of the treatment scheme of the C1498 leukemia mouse model. B is a representative image of in vivo imaging of mice after receiving different treatments (n = 5). C is the appearance and weight statistics of the spleen of mice after receiving different treatments (n = 5). D is a representative image of Wright staining of bone marrow cells of mice after receiving different treatments. EF is H&E analysis of spleen (E) and liver (F) of mice after receiving different treatments. G is the weight change of AML mice during treatment (n = 5). H is the survival curve of AML mice during treatment (n = 10).

[0062] Main experimental materials and equipment:

[0063] Cells: Human AML cells THP-1, HL-60; human CML cells K562; human bone marrow stromal cells HS5. Mouse AML cells C1498. Human O-type red blood cells

[0064] Reagents: P9-PEG2k-DSPE, Pscr-PEG2k-DSPE, IDO1 siRNA, Dox, DiO, DiD, D-luciferin, PBS, calcium ion carrier, CCK8 kit, flow cytometry antibodies (anti-CD11c-APC, anti-CD80-FITC, anti-CD86-PE, anti-CD3-PE, anti-CD8- FITC, anti-Ki67-PE, anti-IFN-γ-PE, anti-PD1-PE and anti-TIM3-PE), Trizol, PCR kit. Equipment: Ultracentrifuge, Zetaview, transmission electron microscope TEM, confocal microscope, fluorescence spectrophotometer, UV spectrophotometer, shaker, small animal live imager, weight scale, PCR instrument.

[0065] Example 1

[0066] This embodiment provides a method for constructing an engineered erythrocyte extracellular vesicle drug delivery system REVs-Dox / siIDO1-PEP (hereinafter referred to as the drug delivery system), and the specific steps are as follows.

[0067] (1) Purify type O erythrocyte-derived extracellular vesicles (REVs) by ultracentrifugation. Characterize the obtained REVs, which are biconcave or cup-shaped vesicles ( Figure 1A ), with a diameter of about 156.7 nm ( Figure 1B ).

[0068] (2) Modifying the purified REVs with the first polypeptide and labeling it as REVs-PEP. The method is to first connect the first polypeptide with DSPE-PEG2k-NHS through the -NH2 group to obtain PEP-PEG2k-DSPE, and then connect PEP-PEG2k-DSPE with REVs to obtain REVs-PEP. The amino acid sequence of the first polypeptide is: SEQ ID NO.1: CDLRSAAVC.

[0069] (3) Drug loading. First, doxorubicin (Dox) was loaded into REVs-PEP by ultrasound to form REVs-Dox-PEP. The Dox loading efficiency was calculated to be 32.01% based on the standard curve ( Figure 1C Then, small interfering RNA (siIDO1) targeting IDO1 was loaded by co-incubation. According to the standard curve of Cy5-siIDO1, it was calculated that approximately 2054 siIDO1 molecules were loaded in each REVs ( Figure 1D and 1E )。 The sequence of the small interfering RNA targeting IDO1 includes SEQ ID NO.3: humansiIDO1 (sense strand, 5′-GGGAAGACCCAAAGGAGUUTT-3′); or, SEQ ID NO.4: mousesiIDO1 (sense strand, 5′-GCAAUAUUGCUGUUCCCUATT-3′).

[0070] Those skilled in the art will appreciate that the small interfering RNA targeting IDO1 can be selected based on the choice of experimental subjects, such as human samples or mouse samples.

[0071] (4) UV-visible spectra showed that compared with unloaded REVs, REVs-Dox / siIDO1-PEP had two more absorption peaks at around 485 nm (Dox) and around 650 nm (Cy5), further supporting the integration of Dox and siIDO1 into REVs.

[0072] (5) Finally, we tested whether REVs-Dox / siIDO1-PEP maintained the typical characteristics of REVs. REVs-Dox / siIDO1-PEP showed a complete morphology similar to that of unloaded REVs, with a negative charge (-9.69 mV); the diameter increased to 171 nm ( Figure 1F In addition, REVs-Dox / siIDO1-PEP did not show obvious size changes when suspended in PBS or serum, and had high storage stability and application stability.

[0073] Example 2

[0074] This example provides in vitro experimental targeting and safety verification of REVs-Dox / siIDO1-PEP constructed in Example 1 on leukemia cells.

[0075] (1) Targeted verification

[0076] Leukemia cells were co-incubated with the DiO-labeled drug delivery system. An experimental group and a control group were set up, the experimental group was REVs-Dox / siIDO1-PEP, and the control group was REVs-Dox / siIDO1-Pscr (Pscr, scrambled PEP). The scrambled PEP used in the control group was called the second polypeptide, and the amino acid sequence of the second polypeptide was: SEQ ID NO.2: RKLRSALKA.

[0077] The results are as follows Figure 2 As shown in the figure, REVs-Dox / siIDO1-PEP was highly internalized by THP-1, C1498 and AML primary cells, all of which highly expressed CLL1. However, REVs-Dox / siIDO1-Pscr showed low internalization in several cells.

[0078] Furthermore, CLL1-negative K562 cells showed low internalization of both REVs-Dox / siIDO1-PEP and REVs-Dox / siIDO1-Pscr, indicating that the first peptide has specific targeting of CLL1. In addition, to further confirm that the uptake of REVs occurs through the specific interaction between REVs-linked PEP and CLL1, pretreatment of leukemia cells with free PEP peptide can eliminate the PEP-mediated increase in REVs uptake. The above experiments prove that PEP-modified REVs are effectively taken up by leukemia cells with high CLL1 expression in vitro.

[0079] (2) Security Verification

[0080] Evaluation of the cytotoxicity of REVs-Dox / siIDO1-PEP: Under the treatment of increasing concentrations of REVs and REVs-Dox / siIDO1-PEP, the cell viability of bone marrow stromal cells HS5 and leukemia cells (THP-1, HL-60 and C1498) was not significantly affected by REVs ( Figure 3 A), but compared with HS5 cells, REVs-Dox / siIDO1-PEP significantly inhibited the cell viability of tumor cells ( Figure 3 B). These data indicate that REVs are not cytotoxic to cells, while REVs-Dox / siIDO1-PEP retains the cytotoxicity of Dox to leukemia cells. After 24 h of treatment with 5 μg / mL REVs-Dox / siIDO1-PEP, approximately 17% of THP-1, 35% of C1498, and 79% of primary AML cells survived, and the killing effect was comparable to that of free Dox at the same concentration ( Figure 3 C). It is worth noting that the control group REVs-Dox / siIDO1-Pscr also showed a slight decrease in cell viability, but the effect was not as obvious as REVs-Dox / siIDO1-PEP ( Figure 3 C).

[0081] The induction of apoptosis of AML cells by each treatment was further quantified. The total apoptosis rate (the sum of early apoptosis and late apoptosis) of leukemia cells treated with REVs-Dox / siIDO1-PEP was significantly higher than that of REVs-Dox / siIDO1-Pscr ( Figure 3 D). At the same time, the gene transfection ability of REVs-Dox / siIDO1-PEP was evaluated. Both REVs-Dox / siIDO1-Pscr and REVs-Dox / siIDO1-PEP could inhibit the expression level of IDO1 in THP-1, C1498 and primary leukemia cells, and REVs-Dox / siIDO1-PEP had a better inhibitory effect ( Figure 3 E-3G).

[0082] In summary, only by accurately and effectively delivering the delivery vector of REVs to leukemia cells can they exert better efficacy in vitro.

[0083] Example 3

[0084] This example provides the efficacy verification of REVs-Dox / siIDO1-PEP constructed in Example 1 in animal experiments (in vivo experiments).

[0085] Based on the in vitro uptake results of REVs-Dox / siIDO1-PEP, the tumor targeting ability of REVs-Dox / siIDO1-PEP was further tested in vivo.

[0086] (1) Luc-GFP-THP-1 and Luc-GFP-C1498 cells were injected into NCG and C57BL / 6 mice through the tail vein, respectively, to establish AML xenograft and allogeneic transplant models. DiD-labeled REVs-Dox / siIDO1-Pscr and REVs-Dox / siIDO1-PEP were injected into AML xenograft and allogeneic transplant mice through the tail vein, respectively, and GFP expression in the bone marrow, liver, spleen, and peripheral blood was detected. + DiD in leukemia cells + Accumulation of REVs.

[0087] The results showed that PEP modification caused DiD in the bone marrow + The percentage of THP-1 cells increased from 13.6% to 48.24%, and DiD in peripheral blood + The percentage of THP-1 cells increased from 11.27% to 54.18%; in addition, the uptake of REVs in spleen and liver was also increased due to the presence of PEP ( Figure 4 A). Similar results were observed in allogeneic transplanted mice ( Figure 5 A).

[0088] (2) Further, we explored whether REVs-Dox / siIDO1-PEP could deliver siIDO1 into leukemia cells and exert a good effect in vivo. qPCR results showed that leukemia cells in mouse bone marrow, spleen and liver IDO1 The expression of Figure 4 B; Figure 5 B).

[0089] In summary, REVs-Dox / siIDO1-PEP can be taken up by leukemia cells in vivo and effectively deliver therapeutic drugs.

[0090] Example 4

[0091] This example verifies that REVs-Dox / siIDO1-PEP constructed in Example 1 can induce immunogenic cell death (ICD) in leukemia cells.

[0092] In this example, free Dox was used as a positive control to verify the effect of REVs-Dox / siIDO1-PEP in inducing ICD, as follows.

[0093] (1) CRT increased on the surface of THP-1 cells treated with REVs-Dox / siIDO1-PEP ( Figure 6 A) Increased release of ATP and HMGB1 in vitro ( Figure 6 B-6C).

[0094] (2) Compared with free Dox treatment, tumor cells treated with REVs-Dox / siIDO1-PEP showed stronger CRT exposure and higher ATP and HMGB1 release ( Figure 6 B-6C), which indicates that REVs-Dox / siIDO1-PEP is precisely delivered into leukemia cells.

[0095] (3) It was further found that CRT exposure, ATP release and HMGB1 secretion of AML primary cells treated with REVs-Dox / siIDO1-PEP were also significantly increased ( Figure 6 E-6F).

[0096] (4) REVs-Dox / siIDO1-PEP treatment also promoted the release of more ATP and HMGB1 by C1498 in vivo ( Figure 6 G-6H).

[0097] (5) AML primary cells treated with REVs-Dox / siIDO1-PEP effectively induced DC maturation in vitro (69.6%), and the induction effect was more significant than that of free Dox ( Figure 6 I-6J).

[0098] In summary, the REVs-Dox / siIDO1-PEP constructed in the present invention has been confirmed to induce immunogenic cell death in leukemia cells, and its effect is more significant than that of free Dox.

[0099] Example 5

[0100] This example verifies that the REVs-Dox / siIDO1-PEP constructed in Example 1 can promote immune activation in vivo, as follows: This example uses the REVs-Dox / siIDO1-PEP constructed in Example 1 to deliver specific small interfering RNA to activate immune activity, and the ability to coordinate ICD to produce composite immune regulation is groundbreaking.

[0101] Experimental groups: PBS treatment group, REVs treatment group, REVs-Dox / siNC-PEP treatment group, REVs- siIDO1-PEP treatment group, and REVs-Dox / siIDO1-PEP treatment group.

[0102] Among them, REVs-Dox / siNC – siNC in PEP (sense strand, 5′-UUCUCCGAACGUGUCACGUTT-3′). The experimental steps are as follows.

[0103] (1) The draining lymph nodes of AML allogeneic transplanted mice were collected after treatment in different experimental groups to detect the maturation of DCs. Compared with the PBS and REVs groups, REVs-Dox / siNC-PEP effectively increased the number of mature DCs to 27.4% ( Figure 7 A). However, REVs-Dox / siIDO1-PEP increased the DC maturation rate to about 30.7% ( Figure 7 A), confirming the effect of the small molecule drug and small interfering RNA synergist proposed in the present invention in promoting DC maturation in vivo, and having a composite immunomodulatory effect.

[0104] (2) CD8 + The proportion of T cells was the highest, indicating that the infiltration of T cells in the bone marrow was effectively promoted ( Figure 7 B).

[0105] (3) After REVs-Dox / siIDO1-PEP administration, tumor-infiltrating CD8 + Enhanced activation and proliferation of T cells ( Figure 7 C-7D). These results demonstrate that REVs-Dox / siIDO1-PEP treatment provides a more comprehensive T cell activation advantage than a single drug delivery system.

[0106] In addition to CD8 + In addition to T cell activation, we further studied the CD8 + T cell exhaustion changes. The upregulation of PD1 (programmed cell death-1) and TIM3 (T-cell immunoglobulin- and mucin-domain-containing-3) is a CD8 + Therefore, the CD8 + Expression of PD1 or TIM3 in T cells.

[0107] (4) REVs-siIDO1-PEP significantly reduced PD1 + CD8 + and TIM3 + CD8 + The percentage of T cells, indicating that siIDO1 alleviated T cell exhaustion ( Figure 7 E-7F). However, REVs-Dox / siIDO1-PEP further led to CD8 + T cell exhaustion rate decreased ( Figure 7 E-7F).

[0108] In summary, the results of this example show that REVs-Dox / siIDO1-PEP can produce a synergistic composite immunomodulatory effect (i.e., activating the immune response while ICD occurs), recruiting more immune cells to infiltrate the bone marrow, thereby reversing the immunosuppressive tumor microenvironment.

[0109] Example 6

[0110] This example verifies the in vivo anti-tumor effect of REVs-Dox / siIDO1-PEP constructed in Example 1.

[0111] Experimental groups: PBS treatment group, REVs treatment group, REVs-Dox / siNC-PEP treatment group, REVs- siIDO1-PEP treatment group, and REVs-Dox / siIDO1-PEP treatment group.

[0112] (1) PBS, REVs, REVs-Dox / siNC-PEP, REVs- siIDO1-PEP and REVs-Dox / siIDO1-PEP with the same drug dose were injected into AML mice through the tail vein. Figure 8 A).

[0113] (2) In vivo imaging was used to monitor the leukemia burden of mice in each experimental group. Figure 8 As shown in B, compared with the PBS group, empty REVs had almost no anti-leukemia effect, and leukemia signals were distributed throughout the mouse body, highly accumulated in the bone marrow, liver, and spleen. Both the REVs-Dox / siNC-PEP and REVs-siIDO1-PEP experimental groups could reduce the leukemia burden to some extent; however, mice treated with the REVs-Dox / siIDO1-PEP group showed significantly lower leukemia burden.

[0114] (3) The spleen is the main extramedullary hematopoietic organ affected by AML, and spleen enlargement is closely related to AML cell infiltration. In this example, after treatment with REVs-Dox / siIDO1-PEP, the spleen enlargement of leukemia mice was alleviated, the spleen volume was significantly reduced, and the spleen weight was significantly reduced ( Figure 8 C). However, spleen enlargement and obvious damage to the red and white pulp were observed in the PBS group and the monotherapy group ( Figure 8 C and 8E). It should be noted that the REVs-()-PEP system can accurately deliver Dox and siIDO1 to leukemia cells, respectively, and both have a certain killing effect on leukemia cells. However, since Dox or siIDO1 alone cannot produce a composite immunomodulatory effect, the single-drug treatment group still has splenomegaly and obvious damage to the red and white pulp.

[0115] (4) Bone marrow cell morphology analysis further showed that the REVs-Dox / siIDO1-PEP treatment group significantly reduced the proliferation of leukemia cells and the number of immature granulocytes decreased ( Figure 8 D).

[0116] (5) The reduction in the infiltration area of ​​the liver of leukemia mice after REVs-Dox / siIDO1-PEP treatment also confirmed the reduction in extramedullary infiltration ( Figure 8 F). In addition, the REVs-Dox / siIDO1-PEP treatment group did not show weight loss, and the life span of leukemia mice was significantly prolonged ( Figure 8 G-8H).

[0117] In summary, REVs-Dox / siIDO1-PEP exhibited effective AML inhibitory effects in animals.

[0118] Example 7

[0119] This embodiment provides a kit for evaluating the therapeutic efficacy of leukemia immunomodulatory drugs and its application, which is used to detect and evaluate the immunomodulatory ability of preclinical drugs in the treatment of leukemia.

[0120] Specifically, the kit is used to evaluate the efficacy of preclinical leukemia immunotherapy drugs, including evaluating whether the drug to be tested induces immunogenic cell death in leukemia cells and / or whether immune activation occurs.

[0121] The kit comprises an engineered erythrocyte extracellular vesicle drug-carrying device, including an erythrocyte extracellular vesicle and a targeting component connected thereto; the targeting component is a polypeptide, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

[0122] The kit is provided with a detection reagent and a control reagent.

[0123] Wherein, the detection reagent is an engineered red blood cell extracellular vesicle drug-carrying device and a drug to be detected.

[0124] Among them, the control reagent is an engineered red blood cell extracellular vesicle drug-carrying device and a control drug; the control drug is a small molecule drug and a small interfering RNA synergist; the small molecule drug includes doxorubicin, and the small interfering RNA is a small interfering RNA targeting IDO1, as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0125] When used, the following steps are involved:

[0126] (1) The test drug and the control drug are loaded into the engineered erythrocyte extracellular vesicle drug loading device respectively, according to the method of Example 1.

[0127] (2) Cultivate leukemia cells with high expression of CLL1.

[0128] (3) The loaded drug to be tested is used in leukemia cells with high expression of CLL1, and the method is referred to Example 4 and Example 5 to detect and evaluate the corresponding indicators (e.g., CRT exposure, ATP and HMGB1 release; CD8 + expression of PD1 or TIM3 in T cells, etc.).

[0129] (4) The control reagent (REVs-Dox / siIDO1-PEP) was used in the same batch of leukemia cells with high CLL1 expression, and the corresponding indicators were detected and evaluated by referring to the method of Examples 4 and 5.

[0130] (5) Compare the indicators detected by the test drug with those detected by the control drug. If the indicators of the test drug are close to or even better than those of the control drug, it is determined that the test drug can be further used in subsequent animal experiments. If the indicators of the test drug and the control drug are far different, it is determined that the test drug does not need to be subjected to subsequent animal experiments for the time being.

[0131] It is understandable that the present invention first proves that REVs-Dox / siIDO1-PEP has a composite immunomodulatory ability, that is, it simultaneously achieves induction of immunogenic cell death and immune activation of leukemia cells, and then confirms in animal experiments that mice treated with REVs-Dox / siIDO1-PEP show significantly lower leukemia load. Therefore, REVs-Dox / siIDO1-PEP can be used as an immunological reagent with proven effects for the evaluation and control of other drugs in the early stages of research and development. When the drug to be tested cannot achieve a good immunomodulatory function in leukemia cells with high expression of CLL1, there is no need to conduct animal experiments, which can save resources and costs.

[0132] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A composite immunomodulator targeting leukemia cells, characterized in that: The leukemia cells are leukemia cells with high expression of CLL1; the composite immunomodulator is composed of a drug delivery system prepared from extracellular vesicles of erythrocytes and contents; the drug delivery system includes extracellular vesicles of erythrocytes and a targeting component connected thereto, the targeting component is a polypeptide, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO.1; the contents are small molecule drugs and small interfering RNA synergists; the small molecule drug is doxorubicin, and the small interfering RNA is a small interfering RNA targeting IDO1; the amino acid sequence of the small interfering RNA targeting IDO1 is a sequence as shown in SEQ ID NO.3 or SEQ ID NO.

4.

2. The composite immunomodulator according to claim 1, characterized in that The dosage form of the composite immunomodulator is injection.

3. The composite immunomodulator according to claim 1, characterized in that The drug loading rate of the small molecule drug loaded in the drug delivery system is greater than 30%.

4. The composite immunomodulator according to claim 1, characterized in that The number of small interfering RNAs loaded in the drug delivery system is greater than 2000.

5. The composite immunomodulator according to claim 1, characterized in that The particle size of the composite immunomodulator ranges from 150nm to 300nm.

6. The composite immunomodulator according to claim 1, characterized in that The erythrocyte extracellular vesicles are derived from type O erythrocytes.

7. The composite immunomodulator according to claim 1, characterized in that The composite immunomodulator has the combined effects of inducing immunogenic cell death and immune activation of leukemia cells.

8. The method for constructing the composite immunomodulator according to any one of claims 1 to 7, characterized in that: The following steps are involved: S01: Extract and purify erythrocyte-derived extracellular vesicles; S02: modifying the erythrocyte-derived extracellular vesicles, connecting the polypeptide fragment to DSPE-PEG2k-NHS via a -NH2 group to obtain PEP-PEG2k-DSPE, and then connecting PEP-PEG2k-DSPE to the erythrocyte-derived extracellular vesicles to obtain REVs-PEP; S03: loading small molecule drugs into the REVs-PEP by ultrasound, and then loading small interfering RNA targeting IDO1 by co-incubation; the amino acid sequence of the small interfering RNA targeting IDO1 is the sequence shown in SEQ ID NO.3 or SEQ ID NO.

4.

9. Use of an engineered erythrocyte extracellular vesicle drug-carrying device in the preparation of a kit for evaluating the therapeutic efficacy of leukemia immunomodulatory drugs, characterized in that: The kit is used to evaluate the efficacy of leukemia immune drugs, and the efficacy includes inducing immunogenic cell death and / or immune activation of leukemia cells; The engineered erythrocyte extracellular vesicle drug-carrying device comprises an erythrocyte extracellular vesicle and a targeting component connected thereto; the targeting component is a polypeptide, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.1; The kit is provided with a detection reagent and a control reagent; The detection reagent is an engineered erythrocyte extracellular vesicle drug-carrying device and a drug to be detected; The control reagent is an engineered erythrocyte extracellular vesicle drug-carrying device and a control drug; the control drug is a small molecule drug and a small interfering RNA synergist; the small molecule drug is doxorubicin, and the small interfering RNA is a small interfering RNA targeting IDO1; the amino acid sequence of the small interfering RNA targeting IDO1 is a sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

Images