Dendritic cell multi-antigen loaded immunotherapy method

Abstract

The invention discloses a dendritic cell multi-antigen loaded immunotherapy method, and a TLR7 agonist compound provided by the invention can strongly activate dendritic cells, significantly enhance the antigen presentation ability of the dendritic cells, and generate a synergistic effect with the dendritic cells loaded with antigens. The immunogenicity of the vaccine is improved, a wider immune cell network can be adjusted, and the anti-tumor immune response is comprehensively enhanced. Through the precisely designed compound structure, the scheme specifically enhances the immune response aiming at the HPV related antigen, and improves the targeting and effectiveness of treatment. The use of the compound is helpful for breaking the immune tolerance of an organism to tumor antigens and exciting stronger immune response. Compared with the pure use of the dendritic cell vaccine, the scheme improves the overall efficacy, possibly reduces the treatment times, and improves the compliance of the patient. Meanwhile, the scheme provides new possibility for individualized immunotherapy of cervical cancer, and precise therapy is realized.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a dendritic cell multi-antigen loaded immunotherapy method. Background Art

[0002] Dendritic cell (DC) vaccine is an innovative cancer immunotherapy method that uses the patient's own dendritic cells to activate immune responses against tumors. As the most powerful antigen-presenting cells in the human body, dendritic cells can effectively capture, process and present antigens, thereby activating T cells and triggering specific immune responses. Cervical cancer is one of the common malignant tumors in women, mainly caused by human papillomavirus (HPV) infection, of which HPV types 16 and 18 are the most common high-risk viruses. The continuous expression of E6 and E7 proteins caused by HPV infection is a key factor in the occurrence and development of cervical cancer. The basic principle of DC vaccine treatment of cervical cancer is to culture the patient's own dendritic cells in vitro and load them with HPV-related antigens (such as E6 and E7 proteins), and then return these treated cells to the patient. These antigen-loaded dendritic cells can activate and amplify specific T cells in vivo, especially cytotoxic T lymphocytes (CTL), thereby launching a strong immune attack against cervical cancer cells expressing HPV antigens. This approach can not only specifically target tumor cells but also may induce long-term immune memory, providing a promising personalized treatment strategy for the treatment of cervical cancer.

[0003] Some scholars have used DC vaccines to treat early cervical cancer and recurrent and refractory cervical cancer patients. The results showed that there was no clinical benefit, and only some patients had serological effects (Santin AD, Bellone S, Palmieri M, et al. HPV16 / 18E7-pulsed dendritic cell vaccination in cervical cancer patients with recurrent disease refractory to standard treatment modalities [J]. Gynecol Oncol, 2006, 100 (3): 469-478. doi: 10.1016 / j.ygyno.2005.09.040.). However, Aipire et al. (Aipire A, Li J, Yuan P, et al. Glycyrrhiza uralensis water extract enhances dendritic cell maturation and antitumor efficacy of HPV dendritic cell-based vaccine [J]. Sci Rep, 2017, 7: 43796. doi: 10.1038 / srep43796.) found that licorice water extract (GUWE) can promote the maturation of DC and increase the production of cytokines in the body. At the same time, it was proved that GUWE induced HPV-specific cell responses and inhibited tumor growth in tumor-bearing mice, which to some extent provides a strategy for the development of DC vaccines. Summary of the invention

[0004] In order to solve or partially solve the problems existing in the related art, the present invention provides a dendritic cell multi-antigen loaded immunotherapy method.

[0005] The first aspect of the present invention provides a pharmaceutical composition for treating cervical cancer, comprising the following components:

[0006] Antigen-loaded dendritic cells and compound LB-27, wherein the compound LB-27 is a compound represented by general formula (I)

[0007]

[0008] Furthermore, the dendritic cells are loaded with antigens including HPV16 E6 / E7 and HPV18 E6 / E7.

[0009] The second aspect of the present application provides a pharmaceutical preparation, comprising the above-mentioned composition and pharmaceutically acceptable excipients.

[0010] The third aspect of the present application provides the use of the above-mentioned composition or the above-mentioned pharmaceutical preparation in the preparation of drugs or vaccines for treating cervical cancer.

[0011] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present invention.

[0012] Beneficial technical effects of the present invention:

[0013] The proposed chemical drug combined with antigen-loaded dendritic cells for the treatment of cervical cancer has significant advantages. The compound in this scheme, as a TLR7 agonist, can strongly activate dendritic cells, significantly enhance their antigen presentation ability, and produce a synergistic effect with antigen-loaded dendritic cells. This not only greatly improves the immunogenicity of the vaccine, but also regulates a wider range of immune cell networks, including T cells, NK cells, etc., to comprehensively enhance the anti-tumor immune response. Through the precisely designed compound structure, this scheme can specifically enhance the immune response to HPV-related antigens, improve the targeting and effectiveness of treatment, and reduce the impact on normal tissues. In addition, the use of compounds helps to break the body's immune tolerance to tumor antigens, so that tumor antigens that may have been ignored can effectively stimulate immune responses. Compared with the use of dendritic cell vaccines alone, this scheme significantly improves the overall efficacy of the vaccine, may reduce the number of treatments required, and improve patient compliance. At the same time, this scheme provides new possibilities for personalized immunotherapy of cervical cancer, and the compound dose and the antigen load of dendritic cells can be adjusted according to the specific conditions of the patient to achieve precise treatment. Finally, the compounds of the present invention are carefully designed to have good biocompatibility and safety, reducing the risk of systemic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 HE staining picture 200 of the blank control group in Experimental Example 1 of the present invention;

[0015] Figure 2 HE staining picture 400 of the blank control group in Experimental Example 1 of the present invention;

[0016] Figure 3 HE staining picture 200 of the single treatment control group in Experimental Example 1 of the present invention;

[0017] Figure 4 HE staining picture 400 of the single treatment group in Experimental Example 1 of the present invention;

[0018] Figure 5 HE staining picture 200 of the combined treatment group in Experimental Example 1 of the present invention;

[0019] Figure 6HE staining picture 400 of the combined treatment group in Experimental Example 1 of the present invention; DETAILED DESCRIPTION

[0020] The optional embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the optional embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0021] The terms used in this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0022] The dendritic cell multi-antigen loaded immunotherapy method of the present invention is described in detail below in conjunction with the accompanying drawings, as follows:

[0023] The first aspect of the present invention provides a pharmaceutical composition for treating cervical cancer, comprising the following components:

[0024] Antigen-loaded dendritic cells and compound LB-27, wherein the compound LB-27 is a compound represented by general formula (I)

[0025]

[0026] (1) Coffee ring structure

[0027] Immunomodulatory activity: The quinoline ring structure in the compound is a common heterocyclic compound with certain immunomodulatory activity. The nitrogen atom on the quinoline ring can participate in the formation of hydrogen bonds, enhancing the interaction with the TLR7 receptor, thereby activating dendritic cells.

[0028] (2) Amino functional group

[0029] Enhanced antigen presentation: The amino functional group (-NH2) plays a key role in this compound. The amino group can enhance the hydrophilicity of the compound, help the compound interact with the dendritic cell membrane, and promote the activation of dendritic cells.

[0030] Stabilizing cytokines: Amino acids also bind to cytokines such as IL-12, increasing their stability in the body and thus enhancing the immune response.

[0031] (3) Carboxyl functional group

[0032] Regulation of immune response: The carboxyl functional group (-COOH) in the compound participates in the formation of ester bonds or amide bonds. The formation of these bonds is related to the interaction with immunomodulatory molecules, thereby regulating the immune response.

[0033] (4) Aliphatic chain

[0034] Increased solubility and bioavailability: Aliphatic chains in the compound (such as isopropyl and ethyl) increase the solubility of the compound and improve its bioavailability in the body, thereby better exerting its immunomodulatory effect.

[0035] (5) Stereoscopic effect of functional groups

[0036] Influence on molecular recognition: The steric effect of functional groups affects the way compounds bind to TLR7 receptors, thereby affecting their ability to activate dendritic cells.

[0037] (6) Electronic effects of functional groups

[0038] Regulation of molecular activity: The electronic effect of functional groups (such as the electron donating effect of amino groups) regulates the overall charge distribution of the compound, affecting its binding to the cell membrane and the biological activity of the molecule.

[0039] In one embodiment of the present application, the dendritic cells are loaded with antigens including HPV16 E6 / E7 and HPV18 E6 / E7.

[0040] In one embodiment of the present application, a pharmaceutical preparation is provided, comprising the above-mentioned composition and pharmaceutically acceptable excipients.

[0041] In one embodiment of the present application, there is provided the use of the above-mentioned composition or the above-mentioned pharmaceutical preparation in the preparation of a drug or vaccine for treating cervical cancer.

[0042] For the purpose of greater clarity, the invention is described in detail through the following examples.

[0043] Embodiment 1:

[0044] Step 1: Synthesis of 4-(isopropylamino)-3-nitroquinoline

[0045] In a dry reaction bottle, 4-chloro-3-nitroquinoline (10 mmol) and isopropylamine (12 mmol) were combined.

[0046] Ethanol (20 mL) was added as solvent.

[0047] The reaction mixture was stirred at room temperature for 2 hours.

[0048] After completion of the reaction, the mixture was poured into ice water, and the precipitate was collected by filtration.

[0049] The precipitate was washed with cold water and dried to give 4-(isopropylamino)-3-nitroquinoline.

[0050] Step 2: Reduction of the nitro group to give 3,4-diamino-N-isopropylquinoline

[0051] 4-(Isopropylamino)-3-nitroquinoline (6 mmol) was dissolved in methanol (50 mL).

[0052] 10% Pd / C catalyst (0.5 g) was added.

[0053] The reaction was stirred at room temperature under a hydrogen atmosphere for 6 hours.

[0054] The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain 3,4-diamino-N-isopropylquinoline.

[0055] Step 3: Introduction of propionyl group to obtain N-(4-(isopropylamino)quinolin-3-yl)propionamide 3,4-Diamino-N-isopropylquinoline (5 mmol) was dissolved in dichloromethane (20 mL).

[0056] Triethylamine (10 mmol) was added as base.

[0057] Propionyl chloride (9 mmol) was slowly added dropwise.

[0058] The reaction was stirred at room temperature for 4 hours.

[0059] After completion of the reaction, the reaction mixture was washed with water, and the organic layer was dried over anhydrous sodium sulfate.

[0060] The solvent was removed by distillation under reduced pressure to obtain N-(4-(isopropylamino)quinolin-3-yl)propionamide. Step 4: Cyclization to obtain the target compound 4-amino-2-ethyl-1-isopropyl-1H-imidazo[4,5-c]quinoline

[0061] N-(4-(Isopropylamino)quinolin-3-yl)propanamide (4 mmol) was dissolved in xylene (20 mL).

[0062] A Lewis acid catalyst (aluminum trichloride, 2 mmol) was added.

[0063] The reaction was heated to reflux for 6 hours.

[0064] After the reaction was complete, the mixture was cooled and poured into ice water.

[0065] The product was extracted with an organic solvent (dichloromethane), and the organic layer was dried over anhydrous sodium sulfate.

[0066] The solvent was removed by distillation under reduced pressure to obtain the target compound. (Compound LB-27)

[0067] 1H NMR (300MHz, DMSO-d6)δ:8.15-8.11(m,1H),7.83-7.79(m,1H),7.70-7.64(m,1H),7.59-7.53(m,1H),6.34(br s,2H,NH2),5.21(s,1H),2.82-2.78(m,2H),1.62(s,3H),1.60(s,3H),1.28(s,3H).

[0068] 13C NMR(75MHz,DMSO-d6)δ:166.7(C),148.0(C),146.6(C),129.0(CH),125.2(CH),124.0(C),1 22.5(CH),122.2(CH),118.2(C),111.9(C),56.9(CH),22.2(2×CH3),21.3(CH2),12.1(CH3).

[0069] Test Example 1

[0070] Materials and Reagents:

[0071] Cervical cancer cell line: HeLa cells ( CCL-2 TM )

[0072] HPV16 E6 / E7 protein (Antigen 1, Abcam, ab233706)

[0073] HPV18 E6 / E7 protein (Antigen 2, Abcam, ab233707)

[0074] Detailed steps:

[0075] 1. Preparation of dendritic cells:

[0076] Monocytes were isolated from peripheral blood of healthy volunteers.

[0077] Monocytes were seeded into 6-well plates at a density of 1×10^6 cells / mL.

[0078] RPMI 1640 medium (containing 10% FBS, 1% penicillin-streptomycin) was added.

[0079] GM-CSF (50 ng / mL) and IL-4 (25 ng / mL) were added.

[0080] The cells were cultured at 37°C, 5% CO2 for 5 days.

[0081] 2. Multi-antigen loading:

[0082] On day 5, HPV16 E6 / E7 protein and HPV18 E6 / E7 protein were mixed at a concentration of 10 μg / mL each.

[0083] The mixed antigens were added to the cultured dendritic cells, and LPS (100 ng / mL) was added as an adjuvant.

[0084] The culture was continued for 48 hours to allow the dendritic cells to be fully loaded with antigens.

[0085] 3. Compound LB-27 treatment:

[0086] Compound LB-27 was dissolved in DMSO to prepare a 10 mM stock solution.

[0087] In the experimental group, compound LB-27 was diluted to a final concentration of 1 μM (adjusted according to the results of the preliminary experiment).

[0088] 4. Construction of cervical cancer mouse model:

[0089] Suspend 1×10^6 HeLa cells in 100 μL PBS.

[0090] The drug was injected subcutaneously into the right inguinal region of BALB / c nude mice.

[0091] Wait for the tumor to grow to about 100 mm 3 (7-10 days).

[0092] 5. Treatment options:

[0093] Combination therapy group:

[0094] Compound LB-27 (10 mg / kg, once a day for 7 consecutive days) was injected intraperitoneally.

[0095] On day 7, 1×10 6 Multiple antigen-loaded dendritic cells.

[0096] Monotherapy group:

[0097] Only on day 7, 1×10 6 Multiple antigen-loaded dendritic cells.

[0098] Blank control group

[0099] No subsequent treatment of the cervical cancer mouse model

[0100] 6. Monitoring and evaluation:

[0101] Tumor size was measured every 3 days using a vernier caliper.

[0102] The mice were sacrificed 28 days after the start of treatment, and tumor tissues and spleens were collected.

[0103] 7. Immune response assessment:

[0104] Prepare spleen cell suspension.

[0105] Flow cytometry was used to detect the proportion of CD4+ and CD8+ T cells.

[0106] IFN-γ and IL-2 levels were detected using ELISA kits.

[0107] Results: a) Flow cytometry detected the proportion of CD4+ and CD8+ T cells:

[0108] Experimental group (chemical drugs combined with multi-antigen loaded dendritic cells treatment group):

[0109] CD4+T cells: 38.5% ± 3.2%

[0110] CD8+ T cells: 29.7% ± 2.8%

[0111] CD4+ / CD8+ ratio: 1.30±0.15

[0112] Control group (multi-antigen loaded dendritic cells treatment group):

[0113] CD4+T cells: 32.1% ± 2.9%

[0114] CD8+ T cells: 24.3% ± 2.5%

[0115] CD4+ / CD8+ ratio: 1.32±0.18

[0116] b) ELISA to detect cytokine levels:

[0117] IFN-γ Level (pg / mL):

[0118] Experimental group: 285.3±42.7

[0119] Control group: 198.6±35.2

[0120] IL-2 Level (pg / mL):

[0121] Experimental group: 76.8±9.5

[0122] Control group: 58.2±7.8

[0123] Result analysis:

[0124] T cell proportion: The CD4+ and CD8+ T cell proportions in the experimental group were higher than those in the control group, indicating that the combined chemotherapy enhanced the proliferation and activation of T cells.

[0125] CD4+ / CD8+ ratio: The ratios in the two groups were similar, indicating that the treatment did not significantly change the balance of T cell subsets.

[0126] IFN-γ level: The IFN-γ level in the experimental group was significantly higher than that in the control group, indicating that the combined chemotherapy enhanced the Th1 immune response and the activity of cytotoxic T cells.

[0127] IL-2 level: The IL-2 level in the experimental group was also higher than that in the control group, indicating that the proliferation and activation of T cells were enhanced.

[0128] 8. Pathological evaluation:

[0129] Tumor tissues were fixed in 10% formalin for 24 h.

[0130] Embed in paraffin and slice (5 μm thick).

[0131] H&E staining was performed.

[0132] Use an optical microscope to observe and take pictures (see the attached picture in the manual).

[0133] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A pharmaceutical composition for treating cervical cancer, characterized in that: Includes the following components: Antigen-loaded dendritic cells and compound LB-27, wherein the compound LB-27 is a compound represented by general formula (I) 2. The pharmaceutical composition according to claim 1, characterized in that Dendritic cells were loaded with antigens including HPV16 E6 / E7 and HPV18 E6 / E7.

3. A pharmaceutical preparation, characterized in that The composition comprises the composition according to claim 1 or 2 and pharmaceutically acceptable excipients.

4. Use of the composition according to claim 1 or 2 or the pharmaceutical preparation according to claim 3 in the preparation of drugs or vaccines for treating cervical cancer.

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