A monophosphoryl lipid A (MPLA)-conjugated glycoprotein antigen Tn anti-tumor vaccine and its application

Abstract

The present invention belongs to the technical field of the development of anti-tumor glycovaccines, and particularly relates to the preparation and application of a fully synthetic anti-tumor glycovaccine in which monophosphoryl lipid A (MPLA) is conjugated with a tumor-associated glycoantigen Tn as an embedded adjuvant. The anti-tumor vaccine described in the present invention is prepared by covalently coupling MPLA with Tn, and has the advantages of clear structure and controllable quality. In addition, the anti-tumor vaccine described in the present invention can improve the immunogenicity of the Tn glycoantigen without the action of an external adjuvant, induce an immune response against the glycoantigen Tn, and thus achieve the purpose of specifically killing tumor cells.

Description

Technical Field

[0001] The present invention relates to the technical fields of chemistry and medicine, and particularly relates to a conjugate of monophosphoryl lipid A (MPLA) and carbohydrate antigen Tn, a preparation method thereof, and an application thereof. Background Art

[0002] Carbohydrate antigen Tn (Thomsen-nouveau, Neu5Acα2-6GalNAcα-O-Ser / Thr) is a tumor-associated carbohydrate antigen (TACA), which is abnormally overexpressed in multiple tumors such as breast cancer, colon cancer, prostate cancer, and lung cancer, and is positively correlated with the deterioration and metastasis of tumor cells, while it is hardly expressed in normal cells. Therefore, Tn is one of the excellent targets for the design of carbohydrate antigen tumor vaccines.

[0003] However, like most TACAs, the immunogenicity of Tn is very poor and it cannot be recognized and presented by the immune system. In order to improve the immunogenicity of Tn carbohydrate antigen, the traditional strategy is to conjugate Tn with carrier proteins (such as KLH, CRM197, and tetanus toxoid TT, etc.) to prepare glycoprotein conjugate vaccines. However, glycoprotein vaccines prepared from Tn antigen and carrier proteins (such as Tn-CRM197) have uncertain conjugation sites, complex components, and need to be used together with adjuvants during immunization. In addition, glycoprotein conjugate vaccines belong to biological products, which are difficult to quality control, have poor stability, and require harsh storage conditions, etc.

[0004]

[0005] Monophosphoryl lipid A (MPLA) is the hydrophobic part of bacterial lipopolysaccharides (LPS), and is an agonist of Toll-like receptor 4 (TLR4), which can targetedly bind to TLR4 and generate an immune response. The present invention uses MPLA as a vaccine carrier and connects it with antigen Tn through an amide bond to prepare a Tn-MPLA fully synthetic vaccine, such as TM01 and TM02. Such vaccines have the advantages of clear chemical structure, single composition, relatively easy quality control, no need for carrier proteins or external adjuvants, etc. At the same time, such fully synthetic vaccines belong to non-protein drugs, have good stability, and simple storage conditions, etc. Summary of the Invention

[0006] The purpose of the present invention is to provide a fully synthetic anti-tumor carbohydrate vaccine in which TLR4 receptor agonist monophosphorylated lipid A (MPLA) is conjugated with carbohydrate antigen Tn. The embedded adjuvant MPLA of the carbohydrate vaccine of the present invention can improve the immunogenicity of carbohydrate antigen Tn, can induce T cell-mediated specific immune responses, produce high concentrations of high-affinity IgG antibodies, and achieve the purpose of specifically killing tumor cells.

[0007] Another object of the present invention is to provide a method for preparing a conjugate of the monophosphorylated lipid A (MPLA) and the sugar antigen Tn.

[0008] Another object of the present invention is to provide the use of the conjugate of the monophosphorylated lipid A (MPLA) and the sugar antigen Tn in the preparation of an anti-tumor vaccine.

[0009] Another object of the present invention is to provide the use of the conjugate of the monophosphorylated lipid A (MPLA) and the sugar antigen Tn in the preparation of an anti-tumor drug.

[0010] The above objects of the present invention are achieved by the following solutions:

[0011] The present invention provides a compound of general formula I, or all possible isomers, pharmaceutically acceptable salts, hydrates or solvates thereof:

[0012] MPLA—Tn;

[0013] Formula I

[0014] Wherein, the monophosphorylated ester A (MPLA) is shown as the following formula:

[0015]

[0016] R1, R2, R3 and R4 are selected from optionally -(CH2) n CH3, -CH2-CH(OH)-(CH2) n CH3, -CH2-CH(O-CO-R5)-(CH2) n CH3, where n is an integer selected from 6-14; R5 is an optionally substituted C 8-14 alkyl; m is an integer selected from 1-10.

[0017] The Tn is shown as the following formula:

[0018]

[0019] X is selected from optionally -NHC(O)-, -CH2-, -NH-, -O-, -C(O)-, -S-, a is an integer selected from 1-8.

[0020] The invention also provides a compound MPLA-Tn of the following general formula, or all possible isomers, pharmaceutically acceptable salts, hydrates or solvates thereof:

[0021]

[0022] In the formula, R1, R2, R3 and R4 are selected from optionally -(CH2)n CH3, -CH2-CH(OH)-(CH2) n CH3, -CH2-CH(O-CO-R5)-(CH2) n CH3, where n is an integer selected from 6 - 14; R5 is an optionally substituted C 8-14 alkyl group; m is an integer selected from 1 - 10; a is any integer from 1 - 8.

[0023] The present invention also provides a compound MPLA-Tn with the following structural formula, or all its possible isomers, pharmaceutically acceptable salts, hydrates or solvates:

[0024]

[0025] The present invention also provides suitable pharmaceutically acceptable salts, hydrates or solvates of the compound shown in Formula I, wherein the pharmaceutically acceptable salts include but are not limited to those formed by reacting with bases such as sodium, potassium, magnesium, calcium, lithium, etc. Some compounds in the present invention may be crystallized or recrystallized with water or organic solvents, and in this case, various solvates may be formed.

[0026] The preparation method of the compound of general formula I is as follows:

[0027]

[0028] The preparation of the above raw material compounds 2 and 3 refers to Patent CN201810845422.7; the preparation of Tn antigen and Tn-CRM197 can be found in the literature (Acta Pharmaceutica Sinica B, 2022, https: / / doi.org / 10.1016 / j.apsb.2022.05.028). The synthetic routes of the representative compounds TM-01 and TM-02 of the compound of general formula I are as above: The MPLA derivatives 2 and 3 are respectively condensed with the amine-containing Tn derivatives to obtain the fully protected compounds 4 and 5, and then hydrogenated under the catalysis of palladium on carbon and palladium hydroxide to obtain the fully synthetic vaccines TM-01 and TM-02.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] (1) The purpose of the present invention is to overcome the deficiencies of the prior art and provide a fully synthetic anti-tumor vaccine of a TLR4 receptor agonist monophosphorylated lipid A (MPLA) conjugated with the sugar antigen Tn. This vaccine has the advantages of clear chemical structure, stable physicochemical properties, simple quality control, and no need for additional adjuvants, etc.

[0031] (2) The preparation method of the conjugate provided by the present invention has a short synthetic route, mild reaction conditions, high yield, and convenient operation, and can be widely applied to industrial preparation.

[0032] (3) The anti-tumor vaccine of the present invention can improve the immunogenicity of Tn glycoantigen without the action of external adjuvants, induce an immune response against the glycoantigen Tn, and thus achieve the purpose of specifically killing tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a diagram for evaluating the IgG immunological activity of glycovaccines TM-01, TM-02 and glycoprotein vaccine Tn-CRM197;

[0034] Figure 2 It is a diagram for evaluating the complement-dependent cytotoxicity of glycovaccines TM-01, TM-02 and glycoprotein vaccine Tn-CRM197. DETAILED DESCRIPTION OF THE INVENTION

[0035] In order to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with embodiments, but the scope of protection of the present invention is not limited thereto.

[0036] Example 1: Preparation of glycovaccine TM-01

[0037] 1) Preparation of compound 2

[0038]

[0039] Dissolve compound 5 (40.0 mg, 16.8 μmol) and Tn derivative (5.3 mg, 20.0 μmol) in DMF, adjust the pH value to 8 with an appropriate amount of N-methylmorpholine under ice bath, warm up to room temperature, react for 20 h, remove DMF under reduced pressure, and separate by TLC silica gel plate chromatography (MeOH / CH2Cl2 1:15, v / v) to obtain compound 4 (25.0 mg, 59.4%). 1 H NMR (400 MHz, CDCl3): δ: 7.30 - 7.11 (m, 30H), 6.81 (s, 1H, NH), 6.73 (t, J = 4.8 Hz, 1H, NH), 5.70 (d, J = 7.8 Hz, 1H, NH), 5.58 - 5.45 (m, 3H, H-3’, 2×NH), 5.19 (t, J = 9.1 Hz, 1H, H-3), 5.08 - 5.03 (m, 3H, H-1’), 4.90 - 4.82 (m, 4H, 2×PhC H 2), 4.52 - 4.42 (m, 7H, 3×PhC H2,H - 4’), 4.38 (d, J = 10.0 Hz, 1H, H - 1), 4.17 - 3.46 (m, 27H), 2.45 - 1.95 (m, 19H), 1.58 - 1.47 (m, 12H), 1.25 (b, 104H, 52×CH2), 0.90 - 0.80 (m, 18H, 3×C H 3). ESI - MS (m / z): 2499.6 [M + H] + 。

[0040] 4) Preparation of glycovaccine TM - 01

[0041]

[0042] Compound 4 (10.0 mg, 4.0 μmol), 9.3 mg of 10% Pd - C and 9.2 mg of Pd(OH)2 were added to 16 mL of CH2Cl2 - MeOH (4:1, v / v). The reaction was carried out at room temperature for 20 h under a hydrogen atmosphere. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain glycovaccine TM - 01 (5.4 mg, 65.8%). 1 1H NMR (400 MHz, CDCl3:CD3OD = 5:1): δ: 5.48 - 5.13 (m, 4H), 4.67 - 4.38 (m, 2H), 4.17 - 3.49 (m, 26H), 2.43 - 1.95 (m, 19H), 1.50 - 1.40 (m, 12H), 1.25 (b, 104H, 52×CH2), 0.90 - 0.80 (m, 18H, 3×C H 3). 31 31P NMR (400 MHz, CDCl3:CD3OD = 3:1): δ: - 2.69. ESI - MS (m / z): 2049.4 [M + H] + 。

[0043] Example 2: Preparation of glycovaccine TM - 02

[0044] 1) Preparation of compound 8

[0045]

[0046] Compound 3 (30.0 mg, 12.6 μmol) and Tn derivative (4.0 mg, 15.2 μmol) were dissolved in DMF. The pH value was adjusted to 8 with an appropriate amount of N - methylmorpholine under an ice bath, and the temperature was raised to room temperature. The reaction was carried out for 20 h. DMF was removed under reduced pressure, and the product was separated by TLC silica gel plate chromatography (MeOH / CH2Cl2 1:15, v / v) to obtain compound 5 (15.6 mg, 49.1%). 11H NMR (400 MHz, CDCl3): δ: 7.34 - 7.11 (m, 30H), 6.81 (s, 1H, NH), 6.73 (t, J = 4.8 Hz, 1H, NH), 5.70 (d, J = 7.8 Hz, 1H, NH), 5.58 - 5.45 (m, 3H, H-3’, 2×NH), 5.16 (t, J = 9.1 Hz, 1H, H-3), 5.02 - 4.99 (m, 3H, H-1’), 4.92 - 4.82 (m, 4H, 2×PhC H 2), 4.52 - 4.42 (m, 7H, 3×PhC H 2, H-4’), 4.38 (d, J = 10.0 Hz, 1H, H-1), 4.17 - 3.46 (m, 27H), 2.45 - 1.95 (m, 19H), 1.58 - 1.47 (m, 12H), 1.25 (b, 108H, 54×CH2), 0.90 - 0.80 (m, 18H, 3×C H 3). ESI-MS (m / z): 2527.6 [M + H] + 。

[0047] 2) Preparation of glycovaccine TM-02

[0048]

[0049] Compound 5 (10.0 mg, 4.0 μmol), 10.8 mg of 10% Pd-C and 10.2 mg of Pd(OH)2 were added to 16 mL of CH2Cl2-MeOH (4:1, v / v), and the reaction was carried out at room temperature for 20 h under a hydrogen atmosphere. The solid was filtered off, and the filtrate was concentrated under reduced pressure to obtain glycovaccine MS-02 (7.1 mg, 85.5%). 1 1H NMR (400 MHz, CDCl3:CD3OD = 5:1): δ: 5.47 - 5.02 (m, 4H), 4.50 - 4.31 (m, 5H), 4.17 - 3.45 (m, 27H), 2.50 - 1.94 (m, 19H), 1.52 - 1.41 (m, 12H), 1.25 (b, 108H, 54×CH2), 0.92 - 0.85 (m, 18H, 3×C H 3). 31 31P NMR (400 MHz, CDCl3:CD3OD = 3:1): δ: -2.70. ESI-MS (m / z): 2077.5 [M + H] + 。

[0050] Experimental Example 1: Immunological Activity Evaluation of Glycovaccines TM-01 and TM-02

[0051] In this experimental example, the conjugates (fully synthetic glycovaccines) prepared in Example 1 and Example 2 were used to immunize mice. Their immunological effects were preliminarily evaluated through ELISA experiments, and the antibody-mediated complement-dependent cytotoxicity (CDC) experiment showed that the antibody serum had the ability to specifically kill tumor cells under the mediation of complement.

[0052] 1) Preparation of the vaccine

[0053] The synthesized conjugate was dissolved in a mixture of DCM-MeOH-H2O (5:5:1, v / v, 2 mL) according to the ratio of conjugate: distearoyl phosphatidylcholine (DSPC): cholesterol = 1:6.5:5, and the solvent was evaporated to dryness. 3.0 mL of 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid (HEPES) buffer (20 mM, pH = 7.5) was added. After sonication for 10 - 20 minutes, the glycovaccines TM-01 and TM-02 were obtained. The concentration of Tn in the two prepared vaccine solutions was 6 μg / 0.1 mL.

[0054] Tn-CRM197 was dissolved in PBS buffer solution, and then aluminum adjuvant was added. After stirring for 30 minutes to mix evenly, a milky white liquid was obtained and adsorbed overnight in a refrigerator at 4°C. The dose of Tn was 1.7 μg / 0.1 mL / mouse.

[0055] 2) Mouse immunization protocol

[0056] Eighteen female BALB / c mice aged 6 - 8 weeks were divided into 3 groups, namely the TM-01 group, the TM-02 group, and the Tn-CRM197 group, with 6 mice in each group. Administration was carried out by subcutaneous injection in the abdomen. A total of 4 subcutaneous injections were given on days 0, 14, 21, and 28, with an injection volume of 0.1 mL per mouse each time. Blood was collected from the orbital cavity on days 0, 21, 27, and 38. The whole blood was placed on ice for 1 h and centrifuged at 4000 revolutions per minute for 15 min at 4°C, and the upper clear serum was taken for ELISA detection and analysis.

[0057] 3) ELISA immunoassay

[0058] Dissolve Tn-HSA in 0.1M carbonate buffer (pH 9.6) to prepare a 2.0 μg / mL solution, add 100.0 μL per well to a 96-well plate, and incubate overnight at 4°C; the next day, incubate in a 37°C incubator for 1 hour; wash the plate with PBST. Add milk blocking solution; add 250.0 μl per well; incubate at room temperature for 1 hour, and wash the plate with PBST. Serum samples from 6 mice in the same group were diluted 300, 900, 2700, 8100, 24300, 72900, 218700, and 656100 times with PBS respectively; add the diluted serum to the 96-well plate at 100.0 μL per well; incubate in a 37°C incubator for 2 hours, and wash the plate 3 times. Add IgG labeled with HRP (diluted 2000 times), add 100.0 μL per well, and incubate at room temperature for 1 hour; wash the plate. Add TMB solution, add 100.0 μL per well, and develop color in the dark at room temperature for 20 minutes. Add 0.5M H2SO4 solution, add 100.0 μL per well. Immediately measure the absorbance with an enzyme-linked immunosorbent assay (ELISA) reader, the detection wavelength is 450 nm, and 570 nm is used as the background wavelength.

[0059] Plot the absorbance (OD) values against the antiserum dilution values and obtain the best-fit line. Use the equation of this line to calculate the dilution value when the OD value reaches 0.2, and calculate the IgG antibody titer according to the reciprocal of the dilution value as Figure 1 shown.

[0060] Experimental results: It can be seen from Figure 1 that both the sugar vaccines TM-01 and TM-02 can induce mice to produce IgG antibodies against the Tn sugar antigen, and the antibody titer increases with the increase in the number of immunizations; the IgG titers produced by the sugar vaccines TM-01 and TM-02 are basically equivalent, but significantly higher than the antibody titer produced by the control glycoprotein vaccine Tn-CRM197, indicating that MPLA, as an endogenous adjuvant, effectively enhances the immunogenicity of the Tn sugar antigen and induces a specific immune response against the Tn sugar antigen.

[0061] 4) Antibody-mediated complement-dependent cytotoxicity (CDC)

[0062] Take breast cancer cells MCF-7 that specifically express the Tn sugar antigen and tumor cells MDA-231 that do not express the Tn sugar antigen and culture them respectively in DMEM medium containing 10% fetal bovine serum (FBS); configure the cells in the logarithmic growth phase into 1.0×10 5A cell suspension with a density of cells / mL was inoculated into a 96-well plate, 100 μL per well, approximately 10,000 cells, and cultured overnight in an incubator. The culture medium was removed, and the cells were washed three times with serum-free MEM culture medium. Then, mouse serum diluted with MEM was added, and the mixture was incubated at 37 °C for 2 hours. The cells were washed three times with serum-free MEM, and a complement solution diluted at a ratio of (1:10) was added, and the cells were cultured at 37 °C for 1 hour. At the same time, low reference (only using serum-free culture medium) and high reference (treated with 5% triton-100) groups were set up. After incubation, the cells were centrifuged, 20 μL of the cell supernatant was taken and diluted to 100 μL with PBS, and 100 μL of LDH cytotoxicity detection reagent was used to develop color for 30 minutes. The absorbance value was detected under the condition of 490 nm, and the cell lysis rate was calculated according to the low reference and high reference wells.

[0063] Experimental results: MCF-7 is a breast cancer cell that overexpresses Tn antigen, and MDA-231 tumor cells that do not express Tn antigen were used as a negative control. Under the same conditions, the antibody sera induced by the glycovaccines TM-01 and TM-02 synthesized in Examples 1 and 2 of the present invention had a significantly higher MCF-7 cell lysis rate mediated by antiserum against mice than the blank serum. There was no cytotoxicity to MDA-231 cells that do not express Tn antigen, indicating that antibodies against Tn glycoprotein antigen can kill tumor cells containing Tn glycoprotein antigen. In particular, the sera of glycovaccines TM-01 and TM-02 had a significantly better toxic effect on MCF-7 than the glycoprotein vaccine Tn-CRM197, which was consistent with the conclusion that the antibody titers against Tn produced by TM-01 and TM-02 were significantly better than those of the glycoprotein vaccine Tn-CRM197. This shows that the glycovaccines TM-01 and TM-02 have more research and development potential than the glycoprotein vaccine Tn-CRM197 and are a very promising anti-tumor vaccine.

[0064] Finally, it should be noted that the above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An MPLA-Tn conjugate, characterized in that, Selected from any one of the following compounds: Or a pharmaceutically acceptable salt thereof.

2. Use of the MPLA-Tn conjugate according to claim 1 in the preparation of a medicament for treating cancer, wherein the cancer is selected from breast cancer.

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