An adjuvant for enhancing the efficacy of dendritic cell vaccines and its application

Abstract

The present invention discloses an adjuvant for enhancing the efficacy of dendritic cell vaccines and its application, belonging to the field of biotechnology. The present invention discloses an adjuvant AOAA for enhancing the efficacy of dendritic cell vaccines, which can significantly enhance the immunogenicity of DCs, upregulate the expression of proinflammatory factors IL-6, IL-12, IL-23, TNFα and co-stimulatory molecules CD80 and CD86 in DCs, and promote DC-mediated CD4 + T cells and CD8 + The ability of T cells to differentiate into IFNγ-secreting effector T cells; moreover, AOAA can be used in the preparation of DC vaccines to enhance the anti-tumor effect of DC vaccines.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to an adjuvant for enhancing the efficacy of dendritic cell vaccines and its application. Background Art

[0002] Cancer is one of the most serious public health problems worldwide, posing a significant threat to human health and life. Traditional cancer therapies, such as surgery, chemotherapy, and radiotherapy, directly target the tumor itself, offering limited efficacy and varying degrees of side effects. Immunotherapy, a recent trend, aims to treat cancer by enhancing or inducing a specific immune response against tumor cells. It boasts a wide range of indications, comprehensive and long-lasting efficacy, and a high safety profile, offering enormous potential for application. Dendritic cell (DC) therapy is the most widely used form of immune cell therapy, primarily due to its ability to efficiently induce effector T cells, as the most powerful antigen-presenting cells, to continuously and stably eliminate cancer cells and prevent cancer recurrence. Numerous studies have demonstrated that cancer treatments such as radiotherapy, checkpoint inhibitors, adoptive T cell therapy, and oncolytic viruses are ineffective in the absence of DCs. Therefore, DC vaccines, alone or in combination with immune checkpoint inhibitors, hold promise as a new breakthrough in cancer immunotherapy. Currently, due to technical and quantitative limitations in obtaining in vivo DCs, DC vaccines are typically derived through in vitro culture and differentiation. Therefore, in the preparation process of DC vaccines, how to improve the immunogenicity of DCs and enhance their ability to induce effector T cells has always been the focus of research by those skilled in the art.

[0003] Therefore, providing an adjuvant for enhancing the efficacy of dendritic cell vaccines and its application is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides an adjuvant for enhancing the efficacy of dendritic cell vaccines and its application.

[0005] Aminooxyacetic acid hemihydrochloride (AOAA) is used as an immune adjuvant in the preparation of dendritic cell (DC) vaccines. AOAA can significantly improve the immunogenicity of DCs and enhance the immunotherapeutic effect of DC vaccines.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] An adjuvant for enhancing the efficacy of dendritic cell vaccines, wherein the adjuvant is aminooxyacetic acid hemihydrochloride.

[0008] Furthermore, aminooxyacetic acid hemihydrochloride is used in the preparation of a preparation for improving the immunogenicity of dendritic cells and enhancing the immunotherapy effect of dendritic cell vaccines.

[0009] Furthermore, aminooxyacetic acid hemihydrochloride is used as an immune adjuvant in the preparation of dendritic cell vaccines.

[0010] Furthermore, during the preparation of the dendritic cell vaccine, the dendritic cells were treated with aminooxyacetic acid hemihydrochloride, and the specific steps were as follows:

[0011] 1) Single bone marrow cells were harvested, resuspended in dendritic cell culture medium containing the cytokines GM-CSF and IL-4, and cultured in a cell culture incubator at 37°C with 5% carbon dioxide for 7 days. During this period, fresh dendritic cell culture medium containing cytokines was replaced based on cell growth. After 7 days, immature dendritic cells with very weak immunogenicity were obtained.

[0012] 2) resuspending the immature dendritic cells obtained in step 1) in fresh dendritic cell culture medium, adding the adjuvant aminooxyacetic acid hemihydrochloride 1 hour before LPS-induced dendritic cell maturation, treating with aminooxyacetic acid hemihydrochloride for 1 hour, and then adding LPS to continue stimulation for 5 hours to obtain mature dendritic cells with strong immunogenicity.

[0013] Furthermore, the final concentrations of GM-CSF and IL-4 are 10 ng / ml and 5 ng / ml, respectively; the final concentration of aminooxyacetic acid hemihydrochloride is 0.1-0.8 mM; and the final concentration of LPS is 100 ng / ml.

[0014] Furthermore, the dendritic cell culture medium is RPMI 1640 culture medium containing fetal bovine serum, double antibiotic penicillin-streptomycin and glutamine; the concentration of the fetal bovine serum is 10% by volume; the final concentrations of penicillin and streptomycin in the double antibiotic are 100 U / ml and 100 g / ml respectively; and the final concentration of glutamine is 2 mM.

[0015] Furthermore, aminooxyacetic acid hemihydrochloride is used in the preparation of a preparation for enhancing the expression of dendritic cell pro-inflammatory factor encoding genes Il6, Il12p40, Il12p35, Il23p19, and Tnfa.

[0016] Furthermore, aminooxyacetic acid hemihydrochloride is used to prepare a preparation for enhancing the expression of CD80 and CD86 in dendritic cells.

[0017] Furthermore, aminooxyacetic acid hemihydrochloride is used in the preparation of a drug that enhances dendritic cell-mediated CD4 + T cells and CD8 + Application of T cells in differentiation preparations into IFNγ-secreting effector T cells.

[0018] Furthermore, aminooxyacetic acid hemihydrochloride is used in the preparation of an enhanced dendritic cell vaccine anti-tumor preparation.

[0019] It can be seen from the above technical solutions that compared with the prior art, the present invention discloses an adjuvant for enhancing the efficacy of dendritic cell vaccines and its application. DC, as the most powerful antigen-presenting cell, takes up processed antigens and presents them to T cells, and then mainly promotes the proliferation and differentiation of T cells by upregulating the expression of co-stimulatory molecules and cytokines. AOAA is a small molecule compound with a chemical formula of C2H5NO3·1 / 2HCl, a molecular weight of 109.3 g / mol, and is easily soluble in water. Experiments have confirmed that AOAA can significantly enhance the immunogenicity of DC, upregulate the expression of pro-inflammatory factors IL-6, IL-12, IL-23, TNFα in DC, and the expression of co-stimulatory molecules CD80 and CD86, and promote DC-mediated CD4 + T cells and CD8 + The ability of T cells to differentiate into IFNγ-secreting effector T cells. Furthermore, AOAA can be used in the preparation of DC vaccines to enhance their anti-tumor effects. This invention provides a method for using the small molecule compound AOAA as an immune adjuvant to enhance the efficacy of DC vaccines, providing new insights into tumor prevention and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0021] Figure 1 The accompanying drawings show the results of fluorescence quantitative PCR detection of the relative expression levels of different cytokines in DCs treated with different concentrations of AOAA according to the present invention after being stimulated by LPS;

[0022] in, Figure 1 a is the fluorescence quantitative PCR detection results of the relative expression of cytokine Il6 in DCs treated with H2O in the control group and 0.1mM, 0.2mM, 0.4mM, and 0.8mM AOAA in the experimental groups after LPS stimulation;

[0023] Figure 1 b is the fluorescence quantitative PCR detection results of the relative expression of cytokine Il12p40 in DCs treated with H2O in the control group and 0.1mM, 0.2mM, 0.4mM, and 0.8mM AOAA in the experimental groups after LPS stimulation;

[0024] Figure 1c is the fluorescence quantitative PCR detection results of the relative expression of cytokine Il12p35 in DCs treated with H2O in the control group and 0.1mM, 0.2mM, 0.4mM, and 0.8mM AOAA in the experimental groups after LPS stimulation;

[0025] Figure 1 d is the fluorescence quantitative PCR detection results of the relative expression of cytokine Il23p19 in DCs treated with H2O in the control group and 0.1mM, 0.2mM, 0.4mM, and 0.8mM AOAA in the experimental groups after LPS stimulation;

[0026] Figure 1 e is the fluorescence quantitative PCR detection results of the relative expression of cytokine Tnfa in DCs treated with H2O in the control group and 0.1mM, 0.2mM, 0.4mM, and 0.8mM AOAA in the experimental groups after LPS stimulation;

[0027] Figure 1 f is the fluorescence quantitative PCR detection results of the relative expression of cytokine Il10 in DCs treated with H2O in the control group and 0.1mM, 0.2mM, 0.4mM, and 0.8mM AOAA in the experimental groups after LPS stimulation;

[0028] Figure 2 The accompanying figures show the flow cytometry results of the expression of the co-stimulatory molecule CD80 in DCs treated with H2O in the control group of the present invention before and after LPS stimulation, and in DCs treated with 0.1 mM, 0.2 mM, 0.4 mM, and 0.8 mM AOAA in the experimental groups after LPS stimulation;

[0029] Figure 3 The accompanying figures show the flow cytometry results of the expression of the co-stimulatory molecule CD86 in DCs treated with H2O in the control group of the present invention before and after LPS stimulation, and in DCs treated with 0.1 mM, 0.2 mM, 0.4 mM, and 0.8 mM AOAA in the experimental groups after LPS stimulation;

[0030] Figure 4 The accompanying figure shows the cell viability of DCs detected by CCK-8 after treatment of DCs with H2O in the control group of the present invention and 0.1 mM, 0.2 mM, 0.4 mM, and 0.8 mM AOAA in the experimental groups for 6 hours;

[0031] Figure 5 The attached figure shows the DCs treated with H2O in the control group and 0.8mM AOAA in the experimental group promoted CD4 + Flow cytometry test results and percentage statistics of T cell differentiation;

[0032] in, Figure 5 a is the DCs treated with H2O in the control group and 0.8 mM AOAA in the experimental group promoted CD4+ Flow cytometry results of T cell differentiation;

[0033] Figure 5 b is the DCs treated with H2O in the control group and 0.8 mM AOAA in the experimental group promoted CD4 + Statistical graph of the percentage of T cell differentiation;

[0034] Figure 6 The attached figure shows the effect of H2O treatment on the DCs in the control group and the experimental group treated with 0.8 mM AOAA on the CD8 + Flow cytometry test results and percentage statistics of T cell differentiation;

[0035] in, Figure 6 a is the DCs treated with H2O in the control group and 0.8 mM AOAA in the experimental group promoted CD8 + Flow cytometry results of T cell differentiation;

[0036] Figure 6 b is the DCs treated with H2O in the control group and 0.8 mM AOAA in the experimental group promoted CD8 + Statistical graph of the percentage of T cell differentiation;

[0037] Figure 7 The accompanying drawings show the growth of subcutaneous tumors and survival curves in mice after in situ injection of DCs treated with H2O in the control group or 0.8 mM AOAA in the experimental group in the subcutaneous tumor model of the present invention;

[0038] in, Figure 7 a is the growth of subcutaneous tumors in mice after orthotopic injection of DCs treated with H2O in the control group or 0.8 mM AOAA in the experimental group in the subcutaneous tumor model;

[0039] Figure 7 b is the survival curve of mice after orthotopic injection of DCs treated with H2O in the control group or 0.8 mM AOAA in the experimental group in the subcutaneous tumor model. DETAILED DESCRIPTION

[0040] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only 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 efforts are within the scope of protection of the present invention.

[0041] The materials and sources used in the following examples are as follows: AOAA (MCE); RPMI 1640 medium (Gibco); fetal bovine serum (Gibco); penicillin-streptomycin (Gibco); glutamine (Gibco); GM-CSF (R&D); IL-4 (Novus); LPS (Sigma); Trizol (Invitrogen); reverse transcription kit (Takara); TB Green fluorescent dye (Takara); anti-CD11c, anti-CD11b, anti-CD80, anti-CD86, anti-CD4, anti-CD8, anti-TCRb, anti-IFNγ, and FixableViability Dye flow cytometry antibodies (Invitrogen); CCK-8 (Biyuntian); Ficoll (GE); naive CD4 + T cell isolation kit (StemCell); naive CD8 + T cell sorting kit (StemCell); PMA (Sigma); Ionomycin (Sigma); GolgiStop (BD); GolgiPlug (BD).

[0042] Example 1 Effects of different concentrations of AOAA on DC cytokine expression

[0043] Experimental methods and grouping: (1) Mouse bone marrow cells were cultured in DC culture medium (RPMI1640 culture medium containing fetal bovine serum, penicillin-streptomycin and glutamine; the fetal bovine serum concentration was 10% by volume; the final concentrations of penicillin and streptomycin in the double antibiotics were 100 U / ml and 100 g / ml, respectively; the final concentration of glutamine was 2 mM) containing cytokines GM-CSF and IL-4 (the final concentrations of GM-CSF and IL-4 were 10 ng / ml and 5 ng / ml, respectively). The cells were placed in a cell culture incubator at 37°C containing 5% carbon dioxide. During the culture, fresh dendritic cell culture medium containing cytokines was replaced according to the growth of the cells. After 7 days, immature DCs with very weak immunogenicity were obtained. (2) Immature DCs were collected in a sterile 50 ml centrifuge tube, centrifuged at 400 g for 5 minutes, and the supernatant was discarded. Fresh DC culture medium was added to resuspend the immature DCs to a concentration of 1×10 6 / ml. (3) The concentration is 1×10 6Immature DCs (1000 cells / ml) were divided into five groups. Group 1 was treated with AOAA-dissolved reagent water (H2O) as the control group. Groups 2 to 5 were experimental groups, with AOAA added at final concentrations of 0.1 mM, 0.2 mM, 0.4 mM, and 0.8 mM, respectively. (4) After the DCs were treated with H2O in the control group and AOAA in the experimental group for 1 hour, LPS at a final concentration of 100 ng / ml was added to the DCs for further stimulation for 5 hours. (5) The cells were collected in 1.5 ml tubes without RNase, centrifuged at 400 g for 5 minutes, and the supernatant was discarded. (6) 200 μl of Trizol was added to each tube to extract RNA, and the genome in the RNA was removed using a genome-free reverse transcription kit and the mRNA was reverse transcribed into a single-strand cDNA. (7) The relative expression levels of cytokine-encoding genes Il6, Il12p35, Il12p40, Il23p19, Tnfa, and Il10 were detected by fluorescence quantitative PCR, with the internal reference gene Gapdh as a reference. The primer sequences used are shown in Table 1.

[0044] Table 1

[0045]

[0046] The results of fluorescence quantitative PCR showed that: (1) Figure 1 As shown in a, compared with the control group, 0.1mM AOAA had no significant effect on the expression of Il6 in DCs, but 0.2mM, 0.4mM and 0.8mM AOAA could significantly enhance the expression level of Il6 in DCs, and the enhancement effect was dose-dependent; (2) Figure 1 As shown in b, compared with the control group, 0.1mM, 0.2mM, 0.4mM and 0.8mM MAOAA can significantly enhance the expression level of Il12p40 in DCs, and the enhancement effect is dose-dependent; (3) Figure 1 As shown in Figure c, compared with the control group, 0.1mM AOAA had no significant effect on the expression of Il12p35 in DCs, but 0.2mM, 0.4mM and 0.8mM AOAA could significantly enhance the expression level of Il12p35 in DCs, and the enhancement effect was dose-dependent; (4) Figure 1 As shown in Figure d, compared with the control group, 0.1mM and 0.2mM AOAA had no significant effect on the expression of Il23p19 in DCs, but 0.4mM and 0.8mM AOAA could significantly enhance the expression level of Il23p19 in DCs, and the enhancement effect was dose-dependent; (5) Figure 1 As shown in Figure e, compared with the control group, 0.1mM, 0.2mM, and 0.4mM AOAA had no significant effect on the expression of Tnfa in DCs, but 0.8mM AOAA could significantly enhance the expression level of Tnfa in DCs; (6) Figure 1As shown in Figure f, compared with the control group, 0.1 mM, 0.2 mM, 0.4 mM and 0.8 mM AOAA had no significant effect on the expression level of Il10 in DCs.

[0047] These data demonstrate that the addition of AOAA prior to LPS-induced DC maturation dose-dependently enhances the expression of the pro-inflammatory cytokines IL-6, IL-12, IL-23, and TNFα encoding genes Il6, Il12p40, Il12p35, Il23p19, and Tnfa in DCs, but does not affect the expression of the anti-inflammatory cytokine IL-10 encoding gene Il10 in DCs. High expression of pro-inflammatory factors can reflect the immunogenicity of DCs, while anti-inflammatory factors can suppress this immunogenicity and enhance immune tolerance. This demonstrates that the immunoadjuvant AOAA of the present invention can enhance the immunogenicity of DCs in a dose-dependent manner.

[0048] Example 2 Effects of different concentrations of AOAA on the expression of DC costimulatory molecules

[0049] Experimental methods and grouping: (1) Bone marrow cells were cultured in DC culture medium containing cytokines GM-CSF and IL-4 for 7 days to obtain immature DCs with very weak immunogenicity. (2) Immature DCs were collected in a sterile 50 ml centrifuge tube, centrifuged at 400 g for 5 minutes, and the supernatant was discarded. Fresh DC culture medium was added to resuspend the immature DCs to a concentration of 1×10 6 / ml. (3) Immature DCs were divided into 6 groups, of which group 1 was treated with AOAA dissolved in reagent water (H2O), and after 1 hour, control reagent PBS was added to serve as the LPS-unstimulated control group; group 2 was treated with AOAA dissolved in reagent water (H2O), and after 1 hour, LPS with a final concentration of 100 ng / ml was added to serve as the LPS-stimulated control group; groups 3 to 6 were experimental groups, and AOAA with a final concentration of 0.1 mM, 0.2 mM, 0.4 mM, and 0.8 mM was added, respectively, and after 1 hour, LPS with a final concentration of 100 ng / ml was added. (4) After 5 hours of treatment with PBS or LPS, the DCs were collected in a 1.5 ml tube, centrifuged at 400 g for 5 minutes, and the supernatant was discarded. The cells were washed once with PBS, resuspended in fresh DC culture medium, and cultured for another 11 hours. (5) The cells were collected in a 1.5 ml tube, centrifuged at 400 g for 5 minutes, and the supernatant was discarded. (6) Prepare a mixture of flow cytometry antibodies including anti-CD11c, anti-CD11b, anti-CD80, anti-CD86 and Fixable Viability Dye, and perform flow cytometry surface staining on the cells. (7) Detect the expression levels of DC costimulatory molecules CD80 and CD86 by flow cytometry. - The cells are living cells, CD11c + CD11b +Double-positive cells were DCs, and the expression levels of CD80 and CD86 were reflected by detecting the mean fluorescence intensity of CD80 and CD86 in DCs.

[0050] The experimental results are as follows Figure 2 As shown, the results of flow cytometry detection showed that the average fluorescence intensity of CD80 in DCs of the LPS-unstimulated control group and the LPS-stimulated control group were 1850 and 3647, respectively, and the average fluorescence intensity of CD80 in DCs treated with 0.1mM, 0.2mM, 0.4mM, and 0.8mM AOAA in the experimental groups were 3830, 4285, 4739, and 5254, respectively.

[0051] The above data show that the expression level of CD80 in mature DCs stimulated by LPS is significantly higher than that in immature DCs without LPS stimulation, and the addition of AOAA before LPS-induced DC maturation can significantly enhance the expression level of CD80 in DCs, and the enhancement effect is dose-dependent.

[0052] The experimental results are as follows Figure 3 As shown, the results of flow cytometry detection showed that the average fluorescence intensity of CD86 in DCs of the LPS-unstimulated control group and the LPS-stimulated control group were 629 and 1079, respectively, and the average fluorescence intensity of CD86 in DCs treated with 0.1mM, 0.2mM, 0.4mM, and 0.8mM AOAA in the experimental groups were 1137, 1268, 1458, and 1725, respectively.

[0053] The above data show that the expression level of CD86 in mature DCs stimulated by LPS is significantly higher than that in immature DCs without LPS stimulation, and the addition of AOAA before LPS-induced DC maturation can significantly enhance the expression level of CD86 in DCs, and the enhancement effect is dose-dependent.

[0054] Example 3 Effects of different concentrations of AOAA on DC cell activity

[0055] Experimental methods and grouping: (1) Bone marrow cells were cultured in DC culture medium containing cytokines GM-CSF and IL-4 for 7 days to obtain immature DCs with very weak immunogenicity. (2) Immature DCs were collected in a sterile 50 ml centrifuge tube, centrifuged at 400 g for 5 minutes, and the supernatant was discarded. Fresh DC culture medium was added to resuspend the immature DCs to a concentration of 1×10 6 / ml. (3) Immature DCs were divided into five groups. Group 1 was treated with AOAA-dissolved reagent water (H2O) as the control group. Groups 2 to 5 were experimental groups, with AOAA added at final concentrations of 0.1mM, 0.2mM, 0.4mM, and 0.8mM, respectively. (4) DCs were treated with H2O in the control group and AOAA in the experimental group for 6 hours. (5) CCK-8 was added to the cells, and after incubation at 37°C for 1 hour, the absorbance at 450nm was measured to calculate cell viability.

[0056] The experimental results are as follows Figure 4 As shown in the figure, compared with the control group, 0.1 mM, 0.2 mM, 0.4 mM and 0.8 mM AOAA had no significant effect on the cell viability of DCs.

[0057] The above data show that the treatment method of DC with the immune adjuvant AOAA in the present invention does not affect the cell viability of DC.

[0058] Example 4 AOAA mediates CD4 + Effects on T cell differentiation

[0059] Experimental method and grouping of DC culture: (1) Bone marrow cells were cultured in DC culture medium containing cytokines GM-CSF and IL-4 for 7 days to obtain immature DC with very weak immunogenicity. (2) Immature DC were divided into two groups. The control group was treated with AOAA dissolved in water (H2O). One hour later, OVA was added to the DC at final concentrations of 5μg / ml and 100ng / ml, respectively. 323-339 (synthesized by Jier Biochemical (Shanghai) Co., Ltd., polypeptide sequence: ISQAVHAAHAEINEAGR; SEQ ID NO. 15) and LPS; the experimental group was treated with 0.8 mM AOAA to treat DCs, and 1 hour later, OVA was added to DCs at final concentrations of 5 μg / ml and 100 ng / ml, respectively. 323-339 and LPS. (3) After 5 hours, DCs were collected and centrifuged at 400g for 5 minutes, after which the supernatant was discarded. The cells were washed once with PBS and resuspended in 1ml of PBS. (4) 1ml of Ficoll was added to the bottom of a 15ml centrifuge tube, and the resuspended cells were carefully layered on the Ficoll. The cells were centrifuged at 800g for 5 minutes, with the acceleration set to the lowest value "1" and the deceleration set to "0". (5) After centrifugation, the live cells in the middle layer were removed, washed once with PBS, and the DCs were resuspended in T cell culture medium (Click's Medium, Irvinesci) and counted.

[0060] DC-CD4 + Experimental methods of T cell co-culture: (1) through the initial CD4 + T cell sorting kit is used to sort naive CD4 cells from OT-II mice. + T cells were sorted and resuspended in T cell culture medium and counted. (2) 2.5×10 4 DCs from the control group or experimental group obtained by the above treatment were added, and 2.5×10 5 Initial CD4 +T cells were co-cultured with DCs. (3) On the 6th day of co-culture, cells were harvested and stimulated with 1 μM ionomycin and 50 ng / mL PMA for 5 hours. GolgiStop and GolgiPlug were then added to prevent extracellular cytokine secretion. (4) CD4 was detected by intracellular staining using flow cytometry. + T cell differentiation.

[0061] The experimental results are as follows Figure 5 a, CD4 + The proportions of T cells secreting IFNγ were 11.3% and 16%, respectively. Figure 5 The statistical results shown in b further showed that CD4 + The proportion of CD4 T cells secreting IFNγ was significantly higher than that of DCs co-cultured with control group. + T cells, and the difference was statistically significant (P < 0.001).

[0062] The above data show that the immune adjuvant AOAA in the present invention can significantly enhance DC-mediated CD4 + The ability of T cells to differentiate into IFNγ-secreting effector T cells.

[0063] Example 5 AOAA mediates CD8 + Effects on T cell differentiation

[0064] Experimental method and grouping of DC culture: (1) Bone marrow cells were cultured in DC culture medium containing cytokines GM-CSF and IL-4 for 7 days to obtain immature DC with very weak immunogenicity. (2) Immature DC were divided into two groups. The control group was treated with AOAA dissolved in water (H2O). One hour later, OVA was added to the DC at a final concentration of 10 ng / ml. 257-264 (Synthesized by Jier Biochemical (Shanghai) Co., Ltd., the polypeptide sequence is: SIINFEKL; SEQ ID NO. 16); The experimental group was treated with 0.8 mM AOAA to treat DCs, and 1 hour later, OVA was added to the DCs at a final concentration of 10 ng / ml. 257-264 (3) After 5 hours, collect DCs, centrifuge at 400g for 5 minutes, discard the supernatant, wash once with PBS, and resuspend the cells in 1ml PBS. (4) Add 1ml Ficoll to the bottom of a 15ml centrifuge tube, carefully spread the resuspended cells on the Ficoll, and centrifuge at 800g for 5 minutes. Set the centrifugation speed to the lowest value "1" and the deceleration speed to "0". (5) After centrifugation, remove the living cells in the middle layer, wash once with PBS, resuspend the DCs in T cell culture medium, and count.

[0065] DC-CD8+ Experimental methods for T cell co-culture: (1) through the initial CD8 + T cell sorting kit was used to sort naive CD8 cells from OT-I mice. + T cells were sorted and resuspended in T cell culture medium and counted. (2) 2.5×10 4 DCs from the control group or experimental group obtained by the above treatment were added, and 2.5×10 5 Initial CD8 + T cells were co-cultured with DCs. (3) On the fourth day of co-culture, cells were harvested and stimulated with 1 μM ionomycin and 50 ng / mL PMA for 5 hours. GolgiStop and GolgiPlug were then added to prevent extracellular cytokine secretion. (4) CD8 T cells were detected by intracellular staining using flow cytometry. + T cell differentiation.

[0066] The experimental results are as follows Figure 6 a) CD8 + The proportions of T cells secreting IFNγ were 18.9% and 32.3%, respectively. Figure 6 The statistical results shown in b further showed that CD8 + The proportion of CD8 T cells secreting IFNγ was significantly higher than that of DCs co-cultured with control group. + T cells, and the difference was statistically significant (P < 0.001).

[0067] The above data show that the immune adjuvant AOAA in the present invention can significantly enhance DC-mediated CD8 + The ability of T cells to differentiate into IFNγ-secreting effector T cells.

[0068] Example 6 Effect of AOAA on the Anti-tumor Effect of DC Vaccine

[0069] 1) Preparation and grouping of DC vaccines: (1) Bone marrow cells were cultured in DC culture medium containing cytokines GM-CSF and IL-4 for 7 days to obtain immature DCs with very weak immunogenicity. (2) The immature DCs were divided into two groups. The control group was treated with AOAA dissolved in H2O. One hour later, OVA was added to the DCs at a final concentration of 10 ng / ml. 257-264 and LPS at a final concentration of 100 ng / ml; the experimental group added 0.8 mM AOAA to treat DCs, and 1 hour later added OVA at a final concentration of 10 ng / ml to DCs 257-264and LPS at a final concentration of 100 ng / ml; (3) After 5 hours, DCs were collected and centrifuged at 400 g for 5 minutes, after which the supernatant was discarded. The cells were washed once with PBS and resuspended in 1 ml of PBS. (4) 1 ml of Ficoll was added to the bottom of a 15 ml centrifuge tube, and the resuspended cells were carefully layered on the Ficoll. The cells were centrifuged at 800 g for 5 minutes, with the acceleration set to the lowest value "1" and the deceleration set to "0". (5) After centrifugation, the live cells in the middle layer were removed, washed once with PBS, and the DCs were resuspended in PBS and counted.

[0070] 2) Experimental methods for establishing a subcutaneous tumor model in mice and injecting DC vaccines in situ: (1) Collect melanoma B16-OVA cells when they are in the logarithmic growth phase, wash them once with PBS, resuspend the melanoma B16-OVA cells in PBS, and count them. (2) Subcutaneously inject wild-type mice with melanoma B16-OVA cells, with 2×10 per mouse injected. 5 (3) One day and four days after tumor cell inoculation, the control group mice were subcutaneously injected with the control group DC vaccine obtained by the above treatment, and the experimental group mice were subcutaneously injected with the experimental group DC vaccine obtained by the above treatment. Each mouse was injected with 2×10 5 (4) Observe the growth of the mouse tumor every day. When a clear black tumor mass appears, measure the tumor size and calculate the tumor area. (5) If the tumor area of ​​the mouse reaches or exceeds 200mm 2 , the mice were immediately euthanized and the survival curves of the mice were calculated.

[0071] The experimental results are as follows Figure 7 As shown in a, compared with the mice inoculated with ordinary DC vaccine in the control group, the subcutaneous tumors of the mice inoculated with AOAA-treated DC vaccine in the experimental group grew more slowly and had smaller tumor sizes, and there was a significant statistical difference in tumor area between the control group and the experimental group (P < 0.001). Figure 7 As shown in Figure b, the survival rate of mice in the control group began to decline on the 16th day and dropped to 0 on the 24th day; the survival rate of mice in the experimental group began to decline on the 20th day, and the survival rate was still 80% on the 24th day, and the survival rate dropped to 50% on the 30th day.

[0072] The above data show that the immune adjuvant AOAA in the present invention can significantly enhance the anti-tumor effect of DC vaccine.

[0073] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The use of aminooxyacetic acid hemihydrochloride in the preparation of an enhanced dendritic cell vaccine anti-tumor preparation, characterized in that: In the preparation of dendritic cell vaccines, dendritic cells are treated with aminooxyacetic acid hemihydrochloride. The specific steps are as follows: 1) Single bone marrow cells were harvested and resuspended in dendritic cell culture medium containing the cytokines GM-CSF and IL-4. The cells were then cultured in a cell culture incubator at 37°C with 5% carbon dioxide for 7 days. During this period, the culture medium containing cytokines was replaced with fresh dendritic cell culture medium based on the cell growth. After 7 days, immature dendritic cells with very weak immunogenicity were obtained. 2) Resuspend the immature dendritic cells obtained in step 1) in fresh dendritic cell culture medium. Add the adjuvant aminooxyacetic acid hemihydrochloride 1 hour before LPS-induced dendritic cell maturation. After aminooxyacetic acid hemihydrochloride treatment for 1 hour, add LPS and continue stimulation for 5 hours to obtain mature dendritic cells with strong immunogenicity. The tumor is melanoma.

2. The use of aminooxyacetic acid hemihydrochloride according to claim 1 in the preparation of an enhanced dendritic cell vaccine anti-tumor preparation, characterized in that: The final concentrations of GM-CSF and IL-4 were 10 ng / ml and 5 ng / ml, respectively; the final concentration of aminooxyacetic acid hemihydrochloride was 0.1 to 0.8 mM; and the final concentration of LPS was 100 ng / ml.

3. The use of aminooxyacetic acid hemihydrochloride according to claim 1 in the preparation of an enhanced dendritic cell vaccine anti-tumor preparation, characterized in that: The dendritic cell culture medium is RPMI 1640 culture medium containing fetal bovine serum, double antibiotic penicillin-streptomycin and glutamine; the concentration of the fetal bovine serum is 10% by volume; the final concentrations of penicillin and streptomycin in the double antibiotic are 100 U / ml and 100 g / ml respectively; the final concentration of glutamine is 2 mM.

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