A monophosphate ester A (MPLA) conjugated sugar antigen STn antitumor vaccine and its application

Abstract

This invention relates to the preparation and application of an antitumor vaccine based on a conjugate of monophosphate ester A (MPLA) and tumor-associated glycoantigen STn, belonging to the field of antitumor glycoantigen vaccine development technology. The antitumor vaccine of this invention can be prepared by chemical methods, possessing the advantages of a well-defined structure, clear composition, and no need for adjuvants. Furthermore, the antitumor vaccine of this invention can induce a stronger immune response against the glycoantigen STn, generating a T-cell-regulated immune response with higher titers, higher affinity, and memory, thereby achieving the purpose of specifically killing tumor cells.

Description

Technical Field

[0001] This invention relates to the fields of chemistry and medicine, and in particular to a conjugate of monophosphate ester A (MPLA) and glycoantigen STn, its preparation method and application. Background Technology

[0002] Tumor-associated glycoantigens (TACAs) are a class of specific glycans that are abnormally overexpressed on the surface of malignant tumor cells. Among them, the glycoantigen STn (Sialyl Thomsen-nouveau, Neu5Acα2-6GalNAcα-O-Ser / Thr) is highly expressed in many tumors such as breast cancer, prostate cancer, colorectal cancer, ovarian cancer, pancreatic cancer, and gastric cancer, but is almost not expressed in normal tissues; STn is also a marker of tumor cell malignancy and metastasis. Therefore, STn is an excellent target for glycoantigen tumor vaccine design. However, like most TACAs, STn has poor immunogenicity and requires the assistance of immunogenic carrier molecules to stimulate T cells and induce a durable antibody response. The traditional strategy is to conjugate the glycoantigen STn to carrier proteins (such as KLH, HSA, CRM197, and BSA) to enhance its immunogenicity. Glycoprotein vaccines obtained by covalently linking STn to the carrier protein CRM197 (STn-CRM197) have been shown to have good immunogenicity. However, glycoprotein vaccines still have disadvantages such as uncertain conjugation sites, unstable conjugation rates, and complex composition (Chemical Science, 2021, 12, 15998-16013).

[0003]

[0004] Monophosphate ester A (MPLA) is the hydrophobic portion of bacterial lipopolysaccharide (LPS) and an agonist of Toll-like receptor 4 (TLR4), capable of targeting and binding to TLR4 to generate an immune response. The polysaccharide portion of LPS extends from the 6′ position of the second glucosamine in the MPLA core. To enhance the binding of MPLA to its receptor TLR4 in the vaccine, this invention links the STn antigen to the 6′ position of the second glucosamine in the MPLA core via an amide bond, while simultaneously increasing the linker chain length, resulting in the novel, fully synthetic vaccines MS-01 and MS-02 of claim 3. Compared to carrier protein glycoprotein conjugate vaccines, these vaccines have advantages such as a well-defined chemical structure, a single component, no need for adjuvants, and relatively simple quality control. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a fully synthetic antitumor vaccine based on a TLR4 receptor agonist monophosphorylated lipid A (MPLA) conjugated sugar antigen STn. This vaccine has the advantages of well-defined chemical structure, stable physicochemical properties, controllable quality control, high specificity, and few side effects.

[0006] Through research, the inventors discovered that linking the glycoantigen STn to the second glucosamine 6′ of the MPLA nucleus enhances the binding of the carrier molecule MPLA to the receptor TLR 4 (Toll-like receptor 4), thereby promoting the recognition and presentation of the STn glycoantigen by the immune system and inducing a T-cell regulated immune response with higher titers, higher affinity, and memory, thus achieving an anti-tumor effect.

[0007] This invention provides compounds of general formula I, or all possible isomers thereof, pharmaceutically acceptable salts, hydrates or solvates:

[0008] MPLA—STn;

[0009] Formula I

[0010] The monophosphate ester A (MPLA) is shown in the following formula:

[0011]

[0012] R1, R2, R3, and R4 are selected from the optional -(CH2) group. n CH3, -CH2-CH(OH)-(CH2) n CH3, -CH2-CH(O-CO-R5)-(CH2) n CH3, where n is an integer selected from 6 to 14; R5 is any C that can be substituted. 8-14 Alkyl; m is an integer selected from 1 to 10.

[0013] The STn is shown in the following formula:

[0014]

[0015] X is selected from any of the following: -NHC(O)-, -CH2-, -NH-, -O-, -C(O)-, -S-. a is an integer selected from 1 to 8.

[0016] The invention also provides compounds of the general formula MPLA-STn, or all possible isomers thereof, pharmaceutically acceptable salts, hydrates or solvates thereof:

[0017]

[0018] In the formula, R1, R2, R3, and R4 are selected from any of the -(CH2) radicals. n CH3, -CH2-CH(OH)-(CH2) n CH3, -CH2-CH(O-CO-R5)-(CH2) n CH3, where n is an integer selected from 6 to 14; R5 is any C that can be substituted. 8-14 Alkyl; m is an integer selected from 1 to 10; a is any integer from 1 to 8.

[0019] This invention also provides a compound with the following structural formula, MPLA-STn, or all possible isomers thereof, pharmaceutically acceptable salts, hydrates, or solvates:

[0020]

[0021] The present invention also provides suitable pharmaceutically acceptable salts, hydrates, or solvates of the compounds shown in Formula I, wherein the pharmaceutically acceptable salts include, but are not limited to, pharmaceutically acceptable salts formed by reactions with bases such as sodium, potassium, magnesium, calcium, lithium, etc. Some compounds of the present invention may be crystallized or recrystallized with water or organic solvents, in which case various solvates may be formed.

[0022] The preparation method of compound I is as follows:

[0023]

[0024] The preparation of raw material compounds 2 and 6 above is based on Chinese Invention Patent (CN109432415A); the preparation of STn antigen, STn-CRM197 and STn-HSA is described in Chemical Science, 2021, 12, 15998-16013. The synthetic routes of representative compounds MS-01 and MS-02 of general formula I are as follows: starting with compound 2, it reacts with succinic anhydride to obtain compound 3; compound 3 reacts with p-nitrophenol to obtain active ester compound 5; compounds 5 and 6 react with STn derivatives containing amine groups to prepare fully protected compounds 7 and 8, and finally hydrogenate to obtain the fully synthetic vaccines MS-01 and MS-02.

[0025] Another object of the present invention is to provide the use of the said conjugate in the preparation of medicaments for the prevention and / or treatment of cancer.

[0026] As a preferred embodiment of the application described in this invention, the cancer is breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, intestinal cancer, renal cell carcinoma, cellular lymphoma, thyroid cancer, brain cancer, stomach cancer, or leukemia.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) In order to overcome the disadvantages of glycoprotein vaccines such as uncertain coupling sites, unstable coupling rates and complex composition, this invention selects STn among tumor-associated glycoantigens (TACAs) with development potential as the research target, and uses lipid A (MPLA) monophosphorylated by TLR4 agonist as an endogenous adjuvant to change the linkage site between STn glycoantigen and MPLA, linking it to STn antigen at the 6′ position of the second glucosamine of MPLA. At the same time, the linkage chain length is increased, enhancing the binding of MPLA in the vaccine to its receptor TLR4, enhancing the immunogenicity of STn glycoantigen, inducing an immune response against STn glycoantigen, and achieving the purpose of specifically killing tumor cells.

[0029] (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 used in industrial preparation. Attached Figure Description

[0030] Figure 1 This is a graph evaluating the IgG immunogenicity of the glycoprotein vaccines MS-01 and MS-02 and the glycoprotein vaccine STn-CRM197.

[0031] Figure 2 This is a graph evaluating complement-dependent cytotoxicity of the glycoprotein vaccines MS-01, MS-02, and STn-CRM197. Detailed Implementation

[0032] To better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0033] Example 1: Preparation of the glycosuriac vaccine MS-01

[0034] 1) Preparation of compound 3

[0035]

[0036] Compound 2 (42 mg, 0.02 mmol) was dissolved in 10 mL of dichloromethane. Succinic anhydride (5 mg, 0.04 mmol) and an appropriate amount of N-methylmorpholine were added to adjust the pH of the reaction solution to 8. After reacting at room temperature for 6 h, the solution was diluted with an appropriate amount of dichloromethane, washed and extracted with saturated NaHCO3, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (methanol:dichloromethane = 1:4) to give compound 3 (36 mg, yield 82%). 1H-NMR(CDCl3,400MHz):7.32-7.12(m,25H),6.75(t,J=4.9Hz,1H,NH),5.73(d,J=7.9Hz,1H,NH'),,5.58-5.5 2(m,2H,H-3',NH'),5.18(t,J=9.1Hz,1H,H-3),5.08-5.00(m,3H,2×Lipid-H,H-1'),4.90-4.80(m,4H,2×PhC H 2), 4.52-4.40(m, 7H, 3×PhC) H 2,H-4'),4.36(d,J=10.0Hz,1H,H-1),3.91(dd,J=1,11.6Hz,1H,H-6),3.90(m,1H,H-2),3.84-3.61( m,6H,H-6,2×H-6',H-5',2×Lipid-H)3.58-3.45(m,6H,H-4,H-5,H-2',OCH3),2.70-2.37(m,10H,COC H 2), 2.31-2.04(m,6H,COC) H 2),1.58-1.47(m,12H),1.25(b,104H,52×CH2),0.90-0.80(m,18H,6×C H 3) ppm. ESI-MS (m / z): 2253.5 [M+H] + .

[0037] 2) Preparation of compound 5

[0038]

[0039] Compound 3 (18 mg, 8 μmol) and p-nitrophenol (5.9 mg, 42 μmol) were dissolved in 5 mL of dichloromethane. EDC·HCl (8.2 mg, 42 μmol) was added under ice bath conditions. The mixture was stirred at room temperature for 6 h, concentrated under reduced pressure, and separated by silica gel column chromatography (methanol: dichloromethane = 1:10) to give compound 5 (15 mg, yield 82%). 1 H-NMR (CDCl3, 400MHz): 8.19 (m, 2H, ArH), 7.35-7.16 (m, 27H), 6.81 (t, J = 4.9Hz, 1H, NH), 5.76 (d, J = 7.9Hz, 1H, NH'),, 5.50-5.53(m,2H,H-3',NH'),5.19(t,J=9.1Hz,1H,H-3),5.08-5.03(m,3H,2×Lipid-H,H-1'),4.90-4.82(m,4H,2×PhCH 2), 4.52-4.42(m, 7H, 3×PhC) H 2,H-4'),4.38(d,J=10.0Hz,1H,H-1),3.98(dd,J=1,11.6Hz,1H,H-6),3.95(dd,J=8.9,10.0Hz,1H,H-2),3.8 5-3.64(m,6H,H-6,2×H-6',H-5',2×Lipid-H)3.58-3.47(m,6H,H-4,H-5,H-2',OCH3),2.70-2.37(m,10H,COC H 2), 2.33-2.05(m, 6H, COC) H 2),1.58-1.47(m,12H),1.25(b,104H,52×CH2),0.90-0.80(m,18H,6×C H 3) ppm. ESI-MS (m / z): 2374.5 [M+H] + .

[0040] 3) Preparation of compound 7

[0041]

[0042] Compound 5 (12 mg, 5 μmol) and STn derivative (4.5 mg, 8 μmol) were dissolved in DMF. The pH was adjusted to 8 with an appropriate amount of N-methylmorpholine (about 6 μL) under ice bath conditions. The mixture was heated to room temperature and reacted for 20 h. DMF was removed under reduced pressure, and the mixture was separated by TLC silica gel plate chromatography (MeOH / CH2Cl2 1:4, v / v) to obtain compound 7 (6 mg, 43.4%). 1 H NMR (400MHz, CDCl3:CD3OD=3:1): δ:7.38-7.14(m,25H),5.50-5.53(t,J=9.1Hz,1H, H-3'),5.19(t,J=9.1Hz,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) H 2,H-4'),4.38(d,J=10.0Hz,1H,H-1),4.07-3.46(m,32H),2.50-2.00(m, 23H),1.58-1.47(m,13H),1.25(b,104H,52×CH2),0.90-0.80(m,18H,3×C H 3) ESI-MS (m / z): 2790.7 [M+H]+ .

[0043] 4) Preparation of the GL-01 sugar vaccine

[0044]

[0045] Compound 7 (6 mg, 2.2 μmol), 5.0 mg 10% Pd-C and 5.2 mg Pd(OH)2 were added to 8 mL CH2Cl2-MeOH (4:1, v / v) and reacted at room temperature under hydrogen atmosphere for 20 h. The solid was removed by filtration and concentrated under reduced pressure to obtain the glycosuric vaccine MS-01 (4.1 mg, 82%). 1 H NMR (400MHz, CDCl3:CD3OD4=3:1): δ:5.50-5.03(m,5H),4.52-4.38(m,3H),4.07-3.40(m,3 2H),2.55-1.95(m,23H),1.50-1.40(m,13H),1.25(b,104H,52×CH2),0.90-0.80(m,18H,3×C H 3). 31 P NMR(400MHz, CDCl3:CD3OD4=3:1):δ:-2.69.ESI-MS(m / z):2340.5[M+H] + .

[0046] Example 2: Preparation of the glycosuriac vaccine MS-02

[0047] 1) Preparation of compound 8

[0048]

[0049] Compound 5 (18 mg, 7.5 μmol) and STn derivative (4.5 mg, 8 μmol) were dissolved in DMF. The pH was adjusted to 8 with an appropriate amount of N-methylmorpholine (about 6 μL) under ice bath conditions. The mixture was heated to room temperature and reacted for 20 h. DMF was removed under reduced pressure, and the mixture was separated by TLC silica gel plate chromatography (MeOH / CH2Cl2 1:4, v / v) to obtain compound 8 (12.7 mg, 59.6%). 1 H NMR (400MHz, CDCl3:CD3OD=3:1): δ:7.33-7.10(m,25H),5.47-5.50(t,J=9.1Hz,1H, H-3'),5.16(t,J=9.1Hz,1H,H-3),5.02-4.99(m,3H,H-1'),4.86-4.80(m,4H,2×PhC H 2), 4.50-4.40 (m, 7H, 3×PhC)H 2,H-4'),4.35(d,J=10.0Hz,1H,H-1),4.05-3.44(m,32H),2.46-1.95(m, 23H),1.55-1.43(m,13H),1.25(b,108H,54×CH2),0.90-0.80(m,18H,3×C H 3) ESI-MS (m / z): 2818.8 [M+H] + .

[0050] 2) Preparation of the glucose vaccine MS-02

[0051]

[0052] Compound 8 (12 mg, 3.5 μmol), 11.0 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). The mixture was reacted at room temperature under hydrogen atmosphere for 20 h. The solid was removed by filtration, and the mixture was concentrated under reduced pressure to obtain the glycosuric vaccine MS-02 (9.1 mg, 91%). 1 H NMR (400MHz, CDCl3:CD3OD4=3:1): δ:5.51-5.06(m,5H),4.53-4.35(m,3H),4.17-3.45(m,3 2H),2.53-1.94(m,23H),1.52-1.41(m,13H),1.26(b,108H,54×CH2),0.90-0.80(m,18H,3×C H 3). 31 P NMR(400MHz, CDCl3:CD3OD4=3:1):δ:-2.69.ESI-MS(m / z):2368.5[M+H] + .

[0053] Experimental Example 1: Evaluation of the immunogenicity of glucose vaccines MS-01 and MS-02

[0054] In this experiment, mice were immunized with the conjugates (fully synthetic sugar vaccines) prepared in Examples 1 and 2. Their immunogenicity was preliminarily evaluated by ELSA assay. Antibody-mediated complement-dependent cytotoxicity (CDC) assay showed that the antibody serum had the ability to specifically kill tumor cells under complement-mediated conditions.

[0055] 1) Vaccine preparation

[0056] The synthesized conjugate was dissolved in a DCM-MeOH-H2O mixture (5:5:1, v / v, 2 mL) at a ratio of conjugate:distearylphosphatidylcholine (DSPC):cholesterol = 1:6.5:5, and the solvent was evaporated. 3.0 mL of hydroxyethylpiperazine ethanethioic acid (HEPES) buffer (20 μm, pH = 7.5) was added. The mixture was sonicated for 10-20 minutes to obtain the glucose vaccines MS-01 and MS-02. The concentration of STn in the two prepared vaccine solutions was 10 μg / 0.1 mL.

[0057] STn-CRM197 was dissolved in PBS buffer solution, and then aluminum adjuvant was added. The mixture was stirred for 30 minutes to obtain a milky white liquid, which was then incubated overnight at 4°C. The STn dose was 2 μg / 0.1 mL / mouse.

[0058] 2) Mouse immunization regimen

[0059] Eighteen female 6-8 week old BALB / c mice were divided into three groups: MS-01, MS-02, and STn-CRM197, with six mice in each group. The drugs were administered subcutaneously via abdominal injection on days 0, 14, 21, and 28, a total of four times, with each injection being 0.1 mL. Blood was collected via orbital sampling on days 0, 21, 27, and 38. Whole blood was incubated on ice for 1 hour, centrifuged at 4000 rpm for 15 minutes at 4°C, and the clear serum supernatant was used for ELISA analysis.

[0060] 3) ELISA Immunoassay

[0061] STn-HSA was dissolved in 0.1M carbonate buffer (pH 9.6) to prepare a 2.0 μg / mL solution. 100.0 μL was added to each well of a 96-well plate and incubated overnight at 4°C. The next day, the plate was incubated at 37°C for one hour. The plate was washed three times with PBST (PBS + 0.05% Tween-20) (300 μL / well / wash). After washing, PBS / 1% BSA was added; 250.0 μL was added to each well. The plate was incubated at room temperature for one hour and washed three times with PBST. Serum samples from six mice in the same group were diluted with PBS at 300, 900, 2700, 8100, 24300, 72900, 218700, and 656100 times, respectively. 100.0 μL of the diluted serum was added to each well of a 96-well plate, with three sub-wells for each dilution gradient. The plates were incubated at 37°C for two hours and washed three times. Add 100.0 μL of HRP (horseradish peroxidase)-labeled IgG (diluted 2000-fold) to each well and incubate at room temperature for one hour; wash the plate three times. Add 100.0 μL of TMB solution to each well and incubate at room temperature in the dark for 20 minutes. Add 100.0 μL of 0.5 M H2SO4 solution to each well. Immediately measure the absorbance using a microplate reader at a detection wavelength of 450 nm, with 570 nm as the background wavelength.

[0062] 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 at which the OD value reaches 0.2, and calculate the IgG antibody titer based on the reciprocal of the dilution value. Figure 1 As shown.

[0063] Experimental results: From Figure 1 It can be seen that, without external adjuvants, both the glycoprotein vaccines MS-01 and MS-02 can induce mice to specifically produce IgG antibodies against the STn glycoprotein antigen, and the effect increases with the number of immunizations. The IgG titer produced by the glycoprotein vaccine MS-02 is better than that produced by the vaccine MS-01, but the IgG antibody titers produced by both are significantly higher than those produced by the control group glycoprotein vaccine STn-CRM197. In particular, the IgG type antibody belongs to the immune response involving T-cells, which can induce immune memory in host cells and promote antibody maturation. This result indicates that the newly discovered vaccines MS-01 and MS-02 are a promising class of anti-tumor vaccines.

[0064] 4) Antibody-mediated complementarity-dependent cytotoxicity (CDC)

[0065] MCF-7 breast cancer cells overexpressing STn glycoantigen and B16-F10 tumor cells not expressing STn glycoantigen were cultured in DMEM medium containing 10% fetal bovine serum (FBS); cells in logarithmic growth phase were configured into 1.0 × 10⁶ cells / cells. 5Cell suspensions at a density of 100 μL / mL were seeded into 96-well plates, approximately 10,000 cells per well, and incubated overnight. After removing the culture medium, the cells were washed three times with serum-free MEM medium, and then diluted with mouse serum (MEM dilution) was added. The plates were incubated at 37°C for 2 hours. After washing three times with serum-free MEM medium, complement solution (1:10 dilution) was added, and the plates were incubated at 37°C for 1 hour. Low-control (serum-free medium only) and high-control (5% Triton-100 treatment) groups were also established. After incubation, the cells were centrifuged, and 20 μL of the cell supernatant was diluted to 100 μL with PBS. The cells were then developed with 100 μL of LDH cytotoxicity assay reagent for 30 minutes. The absorbance of each well was measured at 490 nm, and the cell lysis rate was calculated based on the low-control and high-control wells.

[0066] Experimental results: MCF-7 is a breast cancer cell that overexpresses STn antigen, while B16-F10 tumor cells, which do not express STn antigen, served as a negative control. Figure 2 As shown, the STn antibodies induced in mice by vaccines MS-01, MS-02, and STn-CRM197 showed virtually no toxicity to B16-F10 cells, but significant toxicity to MCF-7 cells. This indicates that antibodies targeting STn glycoantigens can kill tumor cells containing STn glycoantigens. In particular, the serum from glycopeptide vaccines MS-01 and MS-02 showed significantly better toxicity to MCF-7 cells than the glycoprotein vaccine STn-CRM197, with MS-02 showing slightly better performance than MS-01. This suggests that glycopeptide vaccines MS-01 and MS-02 have greater research potential than the glycoprotein vaccine STn-CRM197 and are very promising anti-tumor vaccines.

[0067] 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 changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A conjugate of monophosphate ester A (MPLA) and glycoantigen STn, characterized in that, Its structural formula is shown below: ； ； Or its medicinal salt.

2. The use of the conjugate of monophosphate ester A (MPLA) and glycoantigen STn as described in claim 1 in the preparation of a drug for treating cancer; The cancer in question is breast cancer.

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