Application of crizotinib in inducing tolerance in dendritic cells

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of crizotinib in inducing tolerant dendritic cells (tDCs). This invention provides the application of crizotinib in inducing tolerant dendritic cells. This invention discovers that crizotinib can act as a highly effective inducer of tDCs, significantly inhibiting the activated state of DCs, suppressing their maturation function, reducing their immunostimulatory capacity, and thus inhibiting T cell activation, thereby exerting a therapeutic effect in immune diseases such as GVHD. The inhibitory effects of crizotinib on DCs in this invention include reducing the expression of maturation markers CD80, CD40, and CD86. The crizotinib used in this invention is an FDA-approved ALK / ROS1 inhibitor, primarily used for the treatment of non-small cell lung cancer. This invention discovers its application in immunotherapy, realizing the repurposing of an existing drug, and has significant economic value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of crizotinib in induced tolerance dendritic cells. Background Technology

[0002] Dendritic cells (DCs) are key regulatory cells of the immune system, and their functional state directly determines the direction of the immune response. Tolerogenic dendritic cells (tDCs) are characterized by low expression of co-stimulatory molecules and high secretion of immunosuppressive factors, which can induce T cell tolerance and thus regulate immune homeostasis. Currently, strategies for inducing tDCs are mostly limited to cytokine combinations or gene editing, which suffer from problems such as high cost and poor stability.

[0003] Crizotinib is an FDA-approved ALK / ROS1 inhibitor primarily used for the treatment of non-small cell lung cancer, but its application in inducing tDCs has not been systematically reported. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides the application of crizotinib in induced tolerance dendritic cells. This invention discovers that crizotinib can act as a highly effective inducer of tDCs, significantly inhibiting the activated state of DCs, reducing their immunostimulatory capacity, and further inhibiting T cell activation, thereby exerting a therapeutic effect in immune diseases such as graft-versus-host disease (GVHD).

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides the use of crizotinib in inducing tolerant dendritic cells or in preparing tolerant dendritic cell immune adjuvants.

[0007] Preferably, the induction of tolerant dendritic cells includes inhibiting dendritic cell activation.

[0008] Preferably, the inhibition of dendritic cell activation includes inhibiting the expression of one or more co-stimulatory molecules among CD80, CD86, and CD40.

[0009] The present invention provides a resistant dendritic cell, wherein the method for preparing the resistant dendritic cell includes: co-culturing immature dendritic cells with crizotinib to obtain the resistant dendritic cell.

[0010] Preferably, the concentration of crizotinib in the co-culture system is 5-15 μM.

[0011] Preferably, the immature dendritic cells are obtained from bone marrow cell culture.

[0012] This invention provides the application of the above-described technical solution in the inhibition of T cell activation by tolerating dendritic cells or in the preparation of products for treating diseases related to the suppression of excessive immune responses; the diseases related to the suppression of excessive immune responses include graft-versus-host disease.

[0013] Preferably, the inhibition of T cell activation includes inhibiting the expression of one or more activation markers among CD25, CD69, and CD107a.

[0014] Preferably, the T cells are heterologous T cells.

[0015] Preferably, the treatment for suppressing diseases related to excessive immune responses includes: inhibiting the proliferation of heterologous T cells and / or alleviating the attack of heterologous T cells on host tissues; the host tissues include one or more of the following: skin, liver, spleen, and small intestine.

[0016] This invention provides a resistant dendritic cell (tDC) immune adjuvant, the active ingredient of which is crizotinib; the unit dose of crizotinib in the tDC immune adjuvant is 5-15 μM.

[0017] Beneficial effects:

[0018] This invention discovers that crizotinib can act as a highly effective inducer of tDCs (transmitted digital cells), significantly inhibiting DC activation, suppressing DC maturation, reducing their immunostimulatory capacity, and thereby inhibiting T cell activation, thus playing a therapeutic role in immune diseases such as GVHD. The inhibitory effect of crizotinib on DCs in this invention includes reducing the expression of maturation markers CD80, CD40, and CD86. The crizotinib used in this invention is an FDA-approved ALK / ROS1 inhibitor, primarily used for the treatment of non-small cell lung cancer. This invention discovers its application in immunotherapy, realizing a repurposing of an existing drug, which has significant economic value. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 This illustrates the inhibitory effect of crizotinib on the expression of co-stimulatory molecules in DCs in Example 1 of this invention.

[0021] Figure 2 The results of the experiment on the inhibition of T cell activation markers by crizotinib-tDCs;

[0022] Figure 3 The therapeutic effect of crizotinib-tDCs in a GVHD model (in vivo imaging).

[0023] Figure 4Comparison of H&E staining of organs from different treatment groups. Detailed Implementation

[0024] This invention provides the use of crizotinib in inducing tolerant dendritic cells or in preparing tolerant dendritic cell immune adjuvants.

[0025] In one implementation, the induction of tolerant dendritic cells includes inhibiting dendritic cell activation.

[0026] As one implementation, the inhibition of dendritic cell activation includes inhibiting the expression of one or more co-stimulatory molecules among CD80, CD86, and CD40.

[0027] This invention reveals that crizotinib significantly inhibits dendritic cell activation, reduces the expression of its surface co-stimulatory molecules CD80, CD86, and CD40, and suppresses T cell activation, thereby exerting a therapeutic effect in diseases related to excessive immune activation, such as graft-versus-host disease (GVHD). This invention utilizes the FDA-approved anti-tumor drug crizotinib, achieving a repurposing of an existing drug, and possesses significant clinical translational value and economic potential. This invention provides technical support for the development and treatment of immune diseases such as GVHD based on tDC inducers.

[0028] The present invention provides a resistant dendritic cell, wherein the method for preparing the resistant dendritic cell includes: co-culturing immature dendritic cells with crizotinib to obtain the resistant dendritic cell.

[0029] In one embodiment, the concentration of crizotinib in the co-culture system is 5-15 μM. In another embodiment, the concentration of crizotinib in the co-culture system is 8-12 μM. In yet another embodiment, the concentration of crizotinib in the co-culture system is 10 μM.

[0030] In one embodiment, the immature dendritic cells are obtained from bone marrow cell culture.

[0031] This invention provides the application of the above-described technical solution in the inhibition of T cell activation by tolerating dendritic cells or in the preparation of products for treating diseases related to the suppression of excessive immune responses; the diseases related to the suppression of excessive immune responses include graft-versus-host disease.

[0032] As one implementation, the inhibition of T cell activation includes inhibiting the expression of one or more activation markers among CD25, CD69, and CD107a.

[0033] In one implementation, the T cells are heterologous T cells.

[0034] As one implementation, the treatment for suppressing diseases related to excessive immune responses includes: inhibiting the proliferation of heterologous T cells and / or alleviating the attack of heterologous T cells on host tissues; the host tissues include one or more of the following: skin, liver, spleen, and small intestine.

[0035] Based on the above advantages, this invention provides a tDCs inducer, the active ingredient of which includes crizotinib; the unit dose of crizotinib in the tDCs inducer is 5-15 μM. As one embodiment, the unit dose of crizotinib in the tDCs inducer can be any value within the range of 5-15 μM. As one embodiment, the dosage form of the tDCs inducer includes an injectable formulation. As one embodiment, the tDCs inducer further includes pharmaceutically acceptable excipients. The unit dose described in this invention is the effective unit dose of the tDCs inducer acting on dendritic cells.

[0036] To further illustrate the present invention, the application of crizotinib provided by the present invention in induced tolerance dendritic cells is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0037] The materials used in the following examples and their sources are as follows: Crizotinib (Selleck Chemicals, catalog number S106817); RPMI 1640 medium (Gibco, catalog number 31800022); fetal bovine serum (FBS, PAN-Biotech, catalog number S0615); penicillin-streptomycin (double antibody, Gibco, catalog number 15140122); L-glutamine (Gibco, catalog number 25030081); recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF, PeproTech, catalog number 31503); recombinant mouse IL-4 (rmIL-4, PeproTech, catalog number 21414); lipopolysaccharide (LPS, Sigma, catalog number L3024); flow cytometry antibodies against CD11c, CD80, CD86, CD40, CD25, CD107a, and CD69 were all purchased from BioLegend; D-fluorescein potassium salt (PerkinElmer, catalog number 122799).

[0038] The complete culture medium used in the following examples was RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine, 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 50 μM β-mercaptoethanol, 20 ng / mL rmGM-CSF and 10 ng / mL rmIL-4.

[0039] The method for preparing bone marrow cells is as follows:

[0040] (1) Take the femur and tibia of mice, remove the muscle, disinfect and clean them, rinse the bone marrow cells into a 50mL centrifuge tube with PBS that has been autoclaved and pre-cooled at 4℃, centrifuge at 400g speed for 5 minutes, and remove the supernatant.

[0041] (2) Resuspend the cells with PBS, filter the resuspended cells through a 40μm filter membrane to remove bone fragments and other impurities, centrifuge at 400g for 5 minutes, and remove the supernatant.

[0042] (3) After resuspending the cells in complete culture medium, count them for later use.

[0043] Example 1: Experiment on the effect of crizotinib treatment on the expression of dendritic cell co-stimulatory molecules CD80, CD86, and CD40 after 24 h.

[0044] (1) Mouse bone marrow cells were resuspended in complete culture medium at a concentration of 1.5×106 cells / mL and seeded in 6-well culture plates at 2mL / well. The plates were then cultured in an incubator at 37℃ with 5% carbon dioxide for 7 days. The medium was changed on the 3rd and 5th days of culture. On the 7th day, immature dendritic cells with low immunogenicity were obtained.

[0045] (2) The above immature dendritic cells were resuspended in fresh complete culture medium at a concentration of 1.5×106 cells / mL. Dendritic cells were treated with 10 μM crizotinib for 24 h. The positive control group was treated with 100 ng / mL LPS for 24 h. The negative control group (denoted as Con) consisted of untreated dendritic cells.

[0046] (3) After cell collection, the expression levels of co-stimulatory molecules CD80, CD86, and CD40 on the surface of dendritic cells were detected by flow cytometry staining. CD11c+ cells in living cells are dendritic cells, and the average fluorescence intensity of CD80, CD86, and CD40 in dendritic cells can reflect the expression levels of CD80, CD86, and CD40, respectively.

[0047] Test results as follows Figure 1 As shown in the results, the average positive rates of CD80, CD86, and CD40 in the control group dendritic cells were 60.2%, 48.1%, and 5.6%, respectively, while the average positive rates of CD80, CD86, and CD40 in the dendritic cells treated with 10 μM crizotinib were 12.7%, 11.8%, and 2.6%, respectively.

[0048] The above experimental results indicate that crizotinib can downregulate the expression of co-stimulatory molecules CD80, CD86, and CD40 on the surface of dendritic cells.

[0049] Example 2: Crizotinib-induced tolerance DCs affect heterologous T cell activation

[0050] (1) Mouse bone marrow cells were resuspended in complete culture medium at a concentration of 1.5×106 cells / mL and seeded in 6-well culture plates at 2mL / well. The plates were then cultured in an incubator at 37℃ with 5% carbon dioxide for 7 days. The medium was changed on the 3rd and 5th days of culture. On the 7th day, immature dendritic cells with low immunogenicity were obtained.

[0051] (2) The above immature dendritic cells were resuspended in fresh complete culture medium at a concentration of 1.5×106 cells / mL, and 10 μM crizotinib was added to treat the dendritic cells for 24 h.

[0052] (3) BALB / c mice aged 6-8 weeks were sacrificed by cervical dislocation and their spleens were aseptically separated. The spleens were ground and passed through a 70 μm cell sieve to prepare a single-cell suspension. Red blood cells were lysed using red blood cell lysis buffer. T cells were isolated and purified using a mouse T cell negative sorting kit. The purified T cells were stimulated with phorbolester (PMA) for 12 h, and then the dendritic cells treated with crizotinib in step (2) (denoted as tDCs) were mixed with them and cultured for 72 h (denoted as PMA+tDCs). The dendritic cells not treated with crizotinib were mixed with PMA-stimulated T cells and cultured for 72 h as the PMA group, and the T cells not treated with crizotinib were mixed with PBS-treated T cells and cultured for 72 h as the PBS group. The expression of T cell activation markers CD25, CD107a and CD69 was detected by multicolor flow cytometry.

[0053] Test results as follows Figure 2 As shown in the results, the average positive rates of CD25, CD107a and CD69 in T cells treated with PMA were 12.6%, 11.5% and 71.9%, respectively, while the average positive rates of CD25, CD107a and CD69 in T cells treated with PMA+tDC were 4.46%, 4.88% and 50.8%, respectively.

[0054] The above results indicate that crizotinib-induced resistant DCs can significantly inhibit heterologous T cell activation.

[0055] Example 3: Therapeutic effect of crizotinib-tDCs in a GVHD model (in vivo imaging)

[0056] Thirty BALB / c mice were euthanized by cervical dislocation. Femurs, tibias, and spleens were harvested in a clean bench. Bone marrow was extracted from the femurs and tibias using sterile culture medium. The spleen was ground to prepare a suspension of mononuclear cells from the bone marrow and spleen. Red blood cells were lysed using erythrocyte lysis buffer and resuspended in PBS. C57BL / 6J mice were subjected to total body irradiation (TBI) with 60Co gamma rays at a dose rate of 80 cGy / min, for a total dose of 8.5 Gy. Donor hematopoietic stem cells were infused via the tail vein for transplantation within 4–6 h post-irradiation. Each recipient mouse received 1 × 10⁷ bone marrow cells and 2 × 10⁷ spleen T cells from BALB / c FVB transgenic mice (Fluc.L2G85) (purchased from JAX Laboratories), with a total injection volume of 300 μL per mouse, to establish a GVHD mouse model, ensuring the mice were healthy and met experimental requirements.

[0057] Hematopoietic stem cells were isolated from mouse bone marrow and cultured in vitro to obtain dendritic cells (DCs). Differentiation was induced: DCs were incubated with crizotinib for 24 h to obtain crizotinib-tDCs (denoted as t-DCs), and DCs were incubated with phosphate-buffered saline (PBS) for 24 h to obtain PBS-DCs as control cells (denoted as N-DCs). t-DCs and N-DCs were adoptedively infused via tail vein into the constructed GVHD model mice (infusion dose of 4 × 10⁶ cells / mouse), designated as the t-DCs group and N-DCs group, respectively. Additionally, 300 μL of PBS was infused via tail vein into the constructed GVHD model mice (denoted as the PBS group), and ordinary C57BL / 6J mice (non-GVHD model mice) were used as the Blank group. The expansion and distribution of firefly luciferase reporter gene-labeled T cells in GVHD mice were observed to evaluate the inhibitory effect of crizotinib-tDCs on GVHD in mice.

[0058] Test results as follows Figure 3 As shown, the results indicate that crizotinib-induced tolerant DCs (i.e., crizotinib-tDCs) significantly reduced the proportion of T cell proliferation in GVHD model mice, by more than 5-fold, and the fluorescence intensity of various organs was also significantly reduced. This demonstrates that crizotinib-induced tolerant DCs can significantly inhibit the proliferation and distribution of heterologous T cells in vivo.

[0059] Example 4: Crizotinib-tDCs Organ H&E Staining

[0060] Mouse tissues (skin, liver, spleen, and small intestine) from different groups in Example 3 were fixed in 10% neutral (pH 7.4 ± 0.2) buffered formalin at 25 ± 1°C for 24–48 h, with the fixative volume being 10–20 times the tissue volume. Dehydration was performed using a gradient of ethanol (75%, 85%, 95%, 100%), 1 h per step. The tissues were then sequentially immersed in xylene for 40 minutes each time. After clearing, the tissues were placed in paraffin at 63°C for 50 minutes each time. Sections were cut to a thickness of 4 µm using a microtome, attached to poly-L-lysine-pretreated slides, and baked at 60°C for 1 h. The sections were then sequentially rinsed in xylene (20 minutes × 3 times), a gradient of ethanol (100%, 95%, 85%, 75%), and deionized water for 5 minutes each time. Hematoxylin staining of cell nuclei: Sections were stained with Harris hematoxylin for 5-10 minutes, rinsed with tap water for 10 minutes, differentiated with 1% (v / v) hydrochloric acid ethanol for 30 seconds, and then blued again with bluing solution for 5 minutes. Eosin staining of cytoplasm: Sections were stained with eosin solution for 1-3 minutes. Sections were then sequentially treated with a gradient of ethanol (75%, 85%, 95%, 100%) and xylene, 5 minutes each time. Mounted with neutral resin and cured in the dark for 24 hours. Observation and histological analysis were then performed under a microscope. The microscope was equipped with an image acquisition device and accompanying analysis software.

[0061] Test results as follows Figure 4 As shown in the results, crizotinib-induced tolerant DCs can greatly alleviate the attack of heterologous T cells on various host tissues.

[0062] The above results indicate that crizotinib-induced tolerant DCs can significantly inhibit the occurrence of GVHD in vivo.

[0063] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of crizotinib for the manufacture of a tolerogenic dendritic cell immune adjuvant.

2. Use of crizotinib for the manufacture of an inducer for the in vitro induction of tolerogenic dendritic cells.

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