CN111643661A

CN111643661A - Novel immunologic adjuvant, compound and application thereof

Info

Publication number: CN111643661A
Application number: CN202010516693.5A
Authority: CN
Inventors: 王竹林
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-06-08
Filing date: 2020-06-08
Publication date: 2020-09-11
Also published as: CN113230397B; WO2021249250A1; CN113230397A

Abstract

The invention provides a novel immunologic adjuvant, a compound and application thereof, wherein the immunologic adjuvant is composed of a general formula I. The immune adjuvant forms a tight ligand by using the micromolecule TLR7 stimulant with immune enhancement effect and aluminum hydroxide, and can superpose immune cell activation effect on the basis of keeping the characteristics of the aluminum adjuvant.

Description

Novel immunologic adjuvant, compound and application thereof

Technical Field

The invention relates to the field of immunization and vaccine adjuvants, in particular to a novel immunologic adjuvant, a novel compound and application thereof.

Background

Conventional vaccine adjuvants include inorganic aluminum adjuvants including aluminum hydroxide, aluminum sulfate, aluminum potassium sulfate dodecahydrate (alum), aluminum phosphate, etc., vegetable oil adjuvants, Freund's incomplete adjuvants, complete Freund's adjuvants, etc.; the vegetable oil adjuvant comprises peanut oil emulsion adjuvant, mineral oil, vegetable oil, etc.

The vegetable oil adjuvant and the Freund's adjuvant have adverse reactions such as local stimulation and the like. One of the important roles of traditional aluminum adjuvants in vaccines is to physically adsorb antigen, locally retain and stabilize vaccine components to sufficiently attract immune presenting cells for antigen presenting treatment. The aluminum adjuvant has the advantages of good safety, capability of fixing antigen at an immune position and the like, but the aluminum adjuvant has no activation enhancing effect on immune cells.

Disclosure of Invention

The invention aims to solve the technical problems that the inorganic aluminum series adjuvant lacks the activation and enhancement effect on immune cells and the vegetable oil adjuvant and Freund's adjuvant have adverse reactions such as local stimulation and the like, and provides a novel immunologic adjuvant, a novel compound and application thereof.

The technical scheme for solving the technical problems is to provide a novel immunologic adjuvant, wherein the immunologic adjuvant is composed of a general formula I:

the general formula I consists of phosphorylated micromolecular TLR7 agonist and aluminum hydroxide, wherein n is an integer and is more than or equal to 1 and less than or equal to 100; l is one of a linear alkyl chain and a linear alkoxy chain.

Preferably, L is a linear alkyl chain between 2 and 20 carbon atoms long.

Preferably, L is a linear alkoxy chain with a number of oxygen atoms between 2 and 20.

Preferably, 45. ltoreq. n.ltoreq.55.

Preferably, the structural formula of the immunoadjuvant is:

embodiments of the present invention also provide a compound, which has a structural formula:

the invention also provides an application of the compound in preparing an immunologic adjuvant, wherein the immunologic adjuvant is used as an anti-tumor immune activator.

The invention also provides application of the compound in preparing immunomodulatory drugs, vaccines, immune cell activators or immunopotentiators.

The novel immunologic adjuvant, the compound and the application thereof form a tight ligand through the micromolecule TLR7 excitant with the immunopotentiation effect and aluminum hydroxide, and can superpose the activation effect of immune cells on the basis of keeping the characteristics of the aluminum adjuvant.

Drawings

FIG. 1 is a schematic representation of TLR7 pathway-inducing activity of compounds T-I and T-II;

FIG. 2 is a schematic representation of the tumor-inhibiting effect of the 54 peptide vaccine (Vac);

FIG. 3 is a graph showing the effect of targeted killing by CTLs on each group.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The novel immunological adjuvant AlumT provided by the embodiment of the invention comprises a compound shown in the following general formula I:

wherein L represents a connecting chain. The general formula I consists of a phosphorylated small molecule TLR7 agonist and aluminum hydroxide, wherein the molecular number of the aluminum hydroxide, namely n, is an integer between 1 and 100, preferably an integer between 45 and 55, and most preferably an integer between 50 and 53.

L represents one of a linear alkyl chain or a linear alkoxy chain. In one embodiment of the invention, L is preferably a linear alkyl chain between 2 and 20 carbon atoms long. L may also be a linear alkoxy chain with a number of oxygen atoms between 2 and 20 (PEG chain).

In one embodiment of the invention, a representative of the novel immunological adjuvant, AlumT, consists of the following AlumT-I:

in another embodiment of the invention, a representative of the novel immunological adjuvant, AlumT, consists of the following AlumT-II:

the TLR7 agonist compounds T-I and T-II in AlumT-I and AlumT-II can be synthesized by the following methods:

T-I Synthesis

Namely, 100mg of K-1, 76mg of K-2, 5mg of L-sodium ascorbate and 5mg of CuSO are firstly added₄Dissolve in 100. mu.L water + 400. mu.L of LDMSO, react at room temperature for 5h, and monitor the reaction by LC-MS. After the reaction was completed, purification was performed by HPLC to obtain 50mg of T-I white solid, the yield was 29.7%. ESI-MS: M/z 715.36[ M + H ]]⁺。

Synthesis of T-II

The synthesis method of T-II has the same steps as the synthesis of T-I, namely 100mg of K-1, 76mg of K-3, 5mg of L-sodium ascorbate and 5mg of CuSO are firstly synthesized₄Dissolve in 100. mu.L water + 400. mu.L DMSO, react at room temperature for 5h, and monitor the reaction by LC-MS. After the reaction is finished, purifying by HPLC to obtain a pure product of T-II, ESI-MS, M/z is 708.5[ M + H ]]⁺。

TLR7 of Compounds T-I and T-II activation assays (HEK-Blue) were performed in the following manner^TMDetection)：

Taking HEK-Blue in logarithmic growth phase^TMhTLR7 cells (purchased from InvivoGen), growth medium (Gibco, C11995500BT, Invivo Gen, ant-nr) was discarded, appropriate amounts of 37 ℃ PBS (Hyclone, SH30256.01) were gently rinsed 2 times and PBS was discarded. Adding 2-5mL of PBS (phosphate buffer solution) at 37 ℃, incubating for 1-2 min, scraping cells by using a cell scraper, and then gently blowing and beating the cells to disperse the cells into single cell suspension. Counting the cells and calculating the cell concentration using a hemocytometer using HEK-Blue^TMDecpetion solution (from Invivo Gen) adjusted the cell suspension to 2.5×10⁴Cell plating was performed on 96 well cell culture plates per 180. mu.L well. HEK-Blue was stimulated as designed by addition of concentrations of T-I and T-II (0.2. mu.M, 0.6. mu.M, 1.8. mu.M, 5.5. mu.M, 16.6. mu.M, 50. mu.M)^TMhTLR7 cells, Imiquimod, were positive controls. 3 multiple wells were set for each concentration. Incubating for 6-16 h at 37 ℃ under the condition of 5% carbon dioxide. After the incubation, the absorbance was read at 650nm using a full-wavelength microplate reader (BioTek-Epoch). As shown in fig. 1, the relative induction OD values are relative induction OD values at different concentration values (experimental group mean OD value-negative control group mean OD value)/negative control group mean OD value.

As can be seen from FIG. 1, T-I and T-II significantly activate the TLR7 receptor pathway, belonging to TLR7 agonists.

Example 1 AlumT-I preparation:

10mg (14. mu. mol) of T-I was added to 2.5mL of purified deionized water, and 14. mu.L of a solution containing 0.56mg NaOH was added to form a clear solution. Then, 2mL of Alhydrogel (Invivogen, containing 9.0-11.0mg/mL of aluminum), 1mL of histidine buffer (100mM, pH6.5) and 4.5mL of deionized water were added. The resulting mixture was slowly stirred at room temperature for 1 hour to obtain AlumT-I adjuvant (about 10 mL). Standing at room temperature at 20-25 deg.C for storage. Wherein the mass ratio of T-I to aluminum is 1:2, and the molar ratio is 1: 53. Example 2 preparation of 54 peptide vaccine (Vac) Using peptide 54 peptide of FZD7 protein as antigen

Frizzled7(FZD7) is an antigenic marker of tumor stem cells [ "Frizzled 7as an emeringtarget for cancer therapy". Cell signal.2012apr; 24 (846-51. doi:10.1016/j.cellsig.2011.12.009. "Frizzled-7 as a potential therapeutic target in a colorganic cancer". Neopalasia.2008Jul; 697-705] selecting the main peptide segment 54 as antigen to prepare therapeutic vaccine:

APVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEIC (54 peptide) 10mg is added into 10mL of AlumT-I adjuvant, and the mixture is gently shaken at room temperature to obtain 54 peptide vaccine (Vac).

Example 3 immune anti-tumor experiments with 54 peptide vaccine (Vac vaccine, i.e. 54 peptide + AlumT-I) as therapeutic vaccine

Selecting 4-6 homologous CT26.WT cellsWeek-old BALB/c mice were injected subcutaneously in their backs with 2.0 × 10⁵And C, when the diameter of the tumor reaches 4-5 mm, the CT26.WT cells randomly divide the tumor-bearing mice into a PBS group, an Alum group (aluminum hydroxide group), Alum +54 peptide, T-I +54 peptide and a Vac group (vaccine group, AlumT-I +54 peptide), wherein each group is 10. Both the group injections and the vaccine injections were started, and the Alum group contained Al (1mg/Alhydrogel 100. mu.L), the Alum +54 peptide group (containing Al 1mg/Alhydrogel 100. mu.L plus 54 peptide 100. mu.g), the T-I +54 peptide (containing T-I100. mu.g plus 54 peptide 100. mu.g plus PBS 100. mu.L), and the Vac group (vaccine group, AlumT-I100. mu.L plus 54 peptide 100. mu.g). The injection volume of each group is 100 μ L, and the vaccine is injected on the 8 th, 12 th, 15 th, 19 th and 22 th days after the tumor implantation, 5 times in total, and subcutaneous injection is performed around the tumor. Tumor size volumes were measured periodically for each group of mice. Counting the growth size of each group of tumors on day 36 to calculate the tumor inhibition rate; spleen lymphocytes from each group of mice were extracted for use. The specific effect is shown in fig. 2, and the results shown in fig. 2 indicate that the inhibition rate reaches 90% on day 25, i.e., the Vac vaccine has extremely significant anti-tumor effect.

EXAMPLE 4 tumor cell killing assay (CTL) of effector cells in vitro

On day 25, 3 mean tumor-bearing mice were treated from each group, splenic lymphoid effector cells (using lymphocyte separation medium (Dakewe, Beijing China) and CT26 cells were co-cultured at 50:1 for 4 hours. the killing of effector cells against target cells (CT26) was detected by the LDH method using a non-radioactive cytotoxicity detection kit (Promega, Madison, USA) according to the kit product instructions, and the results are shown in FIG. 3. As can be seen in FIG. 3, the Vac vaccine group had significantly improved targeted killing effects.

Although small molecule TLR7 agonists can act as vaccine adjuvants themselves, their non-specific mobility profile in vivo diminishes the local specificity required as a vaccine. The AlumT adjuvant disclosed by the invention not only maintains the advantages of an aluminum adjuvant, but also overcomes the defect that TLR7 is used as an adjuvant, and superposes the activation effect on immune cells, so that a more beneficial effect is generated.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A novel immunoadjuvant, characterized in that it consists of general formula I:

2. The novel immunoadjuvant of claim 1, wherein L is a linear alkyl chain between 2 and 20 carbon atoms long.

3. The novel immunoadjuvant of claim 1, wherein L is a linear alkoxy chain having an oxygen number of 2 to 20.

4. The novel immunoadjuvant of claim 1, wherein 45. ltoreq. n.ltoreq.55.

5. The novel immunoadjuvant of claim 1, wherein the formula of the immunoadjuvant is:

6. a compound having the formula:

7. use of a compound according to claim 6 for the preparation of an immunoadjuvant for use as an anti-tumour immunoactivator.

8. Use of a compound of claim 6 for the preparation of an immunomodulatory drug, a vaccine, an immune cell activator, or an immunopotentiator.