CN113577266A

CN113577266A - Compound immunopotentiator and preparation method and application thereof

Info

Publication number: CN113577266A
Application number: CN202110975785.4A
Authority: CN
Inventors: 刘芹; 褚雁鸿; 李茹恬; 刘宝瑞
Original assignee: Nanjing Drum Tower Hospital
Current assignee: Nanjing Drum Tower Hospital
Priority date: 2021-08-24
Filing date: 2021-08-24
Publication date: 2021-11-02

Abstract

The invention discloses a composite immunopotentiator and a preparation method and application thereof, wherein the composite immunopotentiator comprises imiquimod suspension and OX40 solution; imiquimod suspensions include imiquimod; solutions of OX40 include OX40 agonist antibodies. The compound immunopotentiator also comprises an auxiliary material, wherein the auxiliary material is a sodium caprylate solution. The imiquimod suspension also comprises a Tween 80 solution and Montanide ISA 51; the Tween 80 solution is prepared by dissolving Tween 80 in normal saline and pure water. The OX40 solution is prepared by dissolving OX40 agonist antibody in physiological saline. The composite immunopotentiator can be used for treating tumors, can enhance the activity of T cells on one hand, and can provide a large amount of antigens for the tumors on the other hand, thereby reducing the immune escape to the maximum extent.

Description

Compound immunopotentiator and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, relates to an immunopotentiator, and particularly relates to a composite immunopotentiator and a preparation method and application thereof.

Background

Immunotherapy has become the most compelling area of development in modern oncology. Based on the immunogenicity of tumors, researchers have used the specificity of the immune system to treat tumors through various channels over the past two decades. T cells play an important role in the immune defense against cancer, with activation or lack of activation being regulated by co-stimulatory receptors (activating receptors) and immune checkpoints (inhibitory receptors). Antibodies that block inhibitory receptors, such as CTLA-4 antibodies, PD-1/PD-L1 antibodies, have made breakthrough progress over the last 25 years.

Agonist antibodies to co-stimulatory receptors (e.g., OX40, etc.) have not consistently achieved good therapeutic efficacy in early trials. In the phase Ib trial of Genentech's OX40 agonist MOXR0916, used in combination with the PDL1 inhibitor atezolizumab, only 2 (4%) of 51 patients receiving treatment had partial responses. Genentech terminated the clinical trial of OX40 agonist at 2019, month 5. In addition, other companies such as the OX40 agonist of GSK have entered clinical trials for four years and are still in the first phase.

The lack of efficacy of OX40 agonists may be due to the lack of optimization of the mode of administration and drug combination.

Disclosure of Invention

The invention discloses a compound immunopotentiator for treating tumors, which comprises an activated anti-OX 40 antibody and imiquimod, wherein the imiquimod can up-regulate the expression of OX40 on T cells, enhance the curative effect of the OX40 antibody and solve the problem of clinical application of the OX40 antibody.

To achieve the above objects, the present invention provides a composite immunopotentiator for tumor treatment, which has the following characteristics: comprises imiquimod suspension and OX40 solution; imiquimod suspensions include imiquimod; solutions of OX40 include OX40 agonist antibodies.

Further, the present invention provides a composite immunopotentiator, which may also have the following characteristics: the auxiliary material is sodium caprylate solution.

Further, the present invention provides a composite immunopotentiator, which may also have the following characteristics: wherein the imiquimod suspension also comprises a Tween 80 solution and Montanide ISA 51; the Tween 80 solution is prepared by dissolving Tween 80 in normal saline and pure water.

Further, the present invention provides a composite immunopotentiator, which may also have the following characteristics: wherein the OX40 solution is prepared by dissolving OX40 agonist antibody in physiological saline.

The invention also provides a preparation method of the composite immunopotentiator, which is characterized in that: the method comprises the following steps:

step one, preparing imiquimod suspension: adding pure water and tween 80 into normal saline to prepare tween 80 solution; adding Montanide ISA51 into Tween 80 solution to obtain mixed emulsion, adding imiquimod powder into the mixed emulsion, mixing, and performing ultrasonic treatment in water bath for 1h to obtain imiquimod suspension;

preparation of OX40 solution: dissolving OX40 agonist antibody in physiological saline to obtain OX40 solution;

preparation of sodium caprylate solution: dissolving sodium caprylate in normal saline to obtain a sodium caprylate solution;

and step two, mixing the imiquimod suspension, the OX40 solution and the sodium caprylate solution, and shaking uniformly to obtain the composite immunopotentiator.

Further, the present invention provides a method for preparing a composite immunopotentiator, which may further have the following characteristics: wherein the concentration of the physiological saline is 0.9 m/v%; in the Tween 80 solution, the volume ratio of normal saline, pure water and Tween 80 is 94.4: 5.4: 0.2; in the imiquimod suspension, the dosage ratio of the Tween 80 solution to the Montanide ISA51 is 1ml to 40 mu l, and the dosage ratio of the mixed emulsion to the imiquimod is 1ml to 2 mg.

Further, the present invention provides a method for preparing a composite immunopotentiator, which may further have the following characteristics: wherein, in the OX40 solution, the dosage ratio of the OX40 agonist antibody to the physiological saline is 10 mg: 1 ml.

Further, the present invention provides a method for preparing a composite immunopotentiator, which may further have the following characteristics: wherein, in the sodium caprylate solution, the dosage ratio of the sodium caprylate to the normal saline is 15 mg: 1.5 ml.

Further, the present invention provides a method for preparing a composite immunopotentiator, which may further have the following characteristics: wherein the volume ratio of the imiquimod suspension, the OX40 solution and the sodium caprylate solution is 1.5: 1: 1.5.

The invention also protects the application of the composite immunopotentiator in preparing tumor treatment medicines.

The invention has the beneficial effects that: the invention provides a compound immunopotentiator for treating tumors, which comprises imiquimod and OX40 excitatory antibody, wherein the imiquimod can up-regulate CD4⁺Expression of OX40 on T cells, further animal experiments showed that the combination of the two exerted a synergistic antitumor effect. The composite immunopotentiator can enhance the activity of T cells on one hand, and can enable tumors to provide a large amount of antigens on the other hand, thereby reducing the immune escape to the maximum extent.

Compared with other common in-situ vaccine strategies such as radiotherapy and radiofrequency ablation, the composite immunopotentiator disclosed by the invention can adopt an intratumoral injection mode, does not depend on special medical equipment, and is more feasible. Compared with the most concerned new antigen vaccine at present, the complex immunopotentiator of the invention has relatively simple implementation process, does not need the fussy steps of second-generation sequencing, new antigen identification, polypeptide synthesis and the like, and can save precious time for patients with diseases in a fast progressive stage. The poor water solubility is the clinical application obstacle of the imiquimod, no injection formulation exists, the imiquimod cream is only externally applied to the surface of the skin in the form of external imiquimod cream at present, and the invention is different from the common imiquimod cream; meanwhile, a certain dosage of Tween 80 and Montanide ISA51 solvents are adopted, so that imiquimod powder can be well dissolved, and a basic guarantee is provided for the imiquimod powder to exert a corresponding curative effect.

Drawings

Figure 1 is imiquimod up-regulating OX40 expression in the tumor microenvironment;

FIG. 2a is a flow chart of a unilateral subcutaneous tumor-bearing mouse model in situ treatment experiment;

FIG. 2b is a graph of the growth of groups of mice in a unilateral subcutaneous tumor-bearing mouse model in situ therapy experiments;

FIG. 2c is a graph of survival for groups of mice in a unilateral subcutaneous tumor-bearing mouse model in situ therapy experiments;

FIG. 3 is a graph of the change in body weight of mice during treatment in a lateral subcutaneous tumor-bearing mouse model;

FIG. 4a is a flow chart of an experiment of in situ treatment of a mouse model loaded with one liver cancer lesion on each side of the abdomen;

FIG. 4b is a graph showing the growth curve (tumor lesion treated) of the in situ treatment experiment of a mouse model with one liver cancer lesion loaded on each side of the abdomen;

FIG. 4c is a graph of growth curves (untreated tumor foci) from in situ treatment experiments in a mouse model loaded with one liver cancer foci on each side of the abdomen;

FIG. 4d is a graph showing the survival rate of the mouse model with one liver cancer lesion loaded on each side of the abdomen;

FIG. 5a is a ratio of central memory T cells and effector memory T cells in the spleen of each group one week after treatment;

FIG. 5b shows spleen cells of mice in NS group and Imiquimod + OX40 agonist antibody group separately from CFSE-labeled H₂₂Incubating liver cancer cells, and adding a ratio chart of dead cells in PI tumor cells after incubation;

FIG. 5c is a graph of the proportion of M2-type macrophages and the level of cytokine IL-10 in each group of tumor microenvironments one week after treatment;

FIG. 6a is a flow chart of an experiment of immune response induced by intratumoral injection of a composite immunopotentiator;

FIG. 6b is a graph showing the tumor growth of each mouse in response to an intratumoral injection of the combined immunopotentiator.

Detailed Description

The present invention is further illustrated by the following specific examples.

The embodiment provides a compound immunopotentiator for treating tumors, which comprises an imiquimod suspension containing imiquimod, an OX40 solution containing OX40 agonist antibodies and an auxiliary material sodium caprylate solution; wherein the imiquimod suspension also comprises a Tween 80 solution and Montanide ISA51, wherein the Tween 80 solution is prepared by dissolving Tween 80 in normal saline and pure water; the OX40 solution was prepared by dissolving OX40 agonist antibody in physiological saline, in this example OX40 agonist antibody was purchased from Xinda corporation under the trade name IBI 101. OX40 agonist antibodies have T cell costimulatory functions. The sodium caprylate solution has the effect of promoting the diffusion of the agent in the tumor tissue.

The preparation method of the compound immunopotentiator comprises the following steps:

step one, preparing imiquimod suspension: adding 5.4ml of pure water and 0.2ml of Tween 80 into 94.4ml of normal saline (0.9 m/v%) to prepare a 0.2% Tween 80 solution; adding 40 μ l Montanide ISA51 into 1ml 0.2% Tween 80 solution, adding 2mg imiquimod powder into 1ml mixed emulsion, mixing, and performing ultrasonic treatment in water bath for 1h (frequency: 80, power: 100, time: 60min) to obtain imiquimod suspension.

Preparation of OX40 solution: 10mg of OX40 agonist antibody was dissolved in 1ml of physiological saline to give an OX40 solution.

Preparation of sodium caprylate solution: 15mg of sodium caprylate was dissolved in 1.5ml of physiological saline to obtain a sodium caprylate solution.

And step two, mixing 1.5ml of imiquimod suspension, 1ml of OX40 solution and 1.5ml of sodium caprylate solution, and shaking uniformly to obtain the composite immunopotentiator.

The application of the compound immunopotentiator in preparing the tumor treatment medicine comprises the following steps:

selection of imiquimod solvent:

preparation of imiquimod suspension

As can be seen from the above table, in a certain amount of tween 80 and Montanide ISA51 solvent, imiquimod can achieve good dissolution, thereby providing a basic guarantee for exerting the corresponding therapeutic effect.

Use of imiquimod and OX40 agonist antibodies in the treatment of tumors:

the relevance of imiquimod and OX40 agonist antibodies was tested. The correlation between TLR7 and OX40 in hepatocellular carcinoma (LIHC) was calculated by an online analysis tool (http:// gepia2.cancer-pku. cn/# correlation), and the results are shown in FIG. 1A, indicating that the expression of OX40 in the tumor microenvironment was positively correlated with the expression of TLR 7. Establishing a unilateral subcutaneous tumor-bearing mouse model, injecting physiological saline (left) or imiquimod (right) once in tumor, excising the tumor after 48h, and detecting CD by flow cytometry³⁺CD⁴⁺Expression of OX40 on T cell subsets, the results are shown in FIG. 1B, which shows tumor-infiltrating CD4 following intratumoral injection of imiquimod⁺There was a 2-fold upregulation of OX40 on the T cell surface. Thus, the results indicate that imiquimod is able to up-regulate the expression of OX40 in the tumor microenvironment.

For a mouse model with unilateral subcutaneous tumor, the compound immunopotentiator has the functions of inhibiting tumor growth and prolonging the life cycle of the mouse. As shown in figure 2a, the in situ vaccine treatment regimen was: day 0 left lower abdomen of Kunming mouse was inoculated with H₂₂Liver cancer cell 2X 10⁶Mice, then on day 5, i.e. when the maximum tumor diameter reached 0.5-0.7cm, treatment with intratumoral injections of a complex immunopotentiator was started, once every other day, for a total of three times. The compound immunopotentiator adopts animal formula, and is calculated by equivalent dose ratio between human and animal according to body surface area, and comprises 20 μ g of imiquimod and 4 μ g of OX40 activated antibody (InVivoMAb anti-mouse OX40(CD134), BioXCell company, Cat. BE 0031); the preparation method comprises the following steps: adding DMSO into imiquimod powder, fully shaking and uniformly mixing to form white emulsion (100mg/ml), adding physiological saline into the white emulsion to dilute to 0.4mg/ml, shaking at the constant temperature of 37 ℃ for 1-2 hours to form uniform and stable small particle suspension, and adding OX40 activated antibody (the final concentration is 0.08 mg/ml); 50 μ l of the composite immunopotentiator was injected into mouse tumor tissue using a 1ml syringe.

The growth curves for each group of mice are shown in figure 2b, and the growth curves represent the average tumor volume for each group. Grouping mice: saline treated group (NS), Imiquimod monotherapy group (Imiquimod), OX40 antibody monotherapy group (OX40 agonistic antibody), Imiquimod and OX40 antibody combination treatment group (Imiquimod + OX40 agonistic antibody), ten mice per group. The survival curves (n ═ 10) for each group of mice are shown in fig. 2 c. The results show that tumor growth was very rapid in NS treated mice, with only a slight delay in tumor growth caused by either imiquimod or OX 40-agonist antibody alone, but the combined injection of the two immunopotentiators resulted in complete regression of some of the mice tumors and a significant prolongation of survival.

The safety of the compound immunopotentiator injected into tumor is high. The body weights of the mice were recorded during treatment in a unilateral subcutaneous tumor-bearing mouse model and the changes in body weight of the mice are shown in fig. 3, which shows that the average body weight of each group of mice has a similar pattern of change.

After the compound immunopotentiator is injected into the tumor on one side of the mouse loaded with one liver cancer focus on each side of the abdomen, the growth of the injected tumor focus and the growth of the distant tumor are both obviously inhibited. As shown in fig. 4a, the in situ vaccine treatment regimen was: inoculating H to left lower abdomen and right lower abdomen of Kunming mouse respectively on day 0₂₂Liver cancer cell 2X 10⁶Mice, then on day 5, i.e. when the maximum tumor diameter reached 0.5-0.7cm, treatment of the left lower abdominal tumor with intratumoral injection of a complex immunopotentiator (20. mu.g imiquimod and 4. mu.g of OX40 agonist antibody) was initiated, once every other day, for a total of three times. The growth curves for each group of mice are shown in figures 4b and 4c, the growth curves representing the average volume of treated tumor lesions (3b) and untreated tumor lesions (3 c). Grouping mice: saline treated group (NS), Imiquimod monotherapy group (Imiquimod), OX40 antibody monotherapy group (OX40 agonistic antibody), Imiquimod and OX40 antibody combination treatment group (Imiquimod + OX40 agonistic antibody), six mice per group. The survival curves of the groups of mice are shown in fig. 4 d. The results show that tumors in NS treated mice show progressive rapid growth at both treated and untreated sites; tumor growth trends were slightly slower with treatment with imiquimod or OX40 agonist antibody alone;but the combined injection of the two immunopotentiators resulted in complete regression of local and distant tumors in nearly half of the mice; consistent with the time required to initiate and enhance the immune response, the regression kinetics of the two tumors differ, with untreated tumors regressing at a rate several days slower than localized tumors; the group of Imiquimod + OX40 agonist antibodies survived significantly longer than any other group.

The tumor immunity microenvironment is activated by injecting the compound immunopotentiator in the tumor. One week after completion of the last treatment, spleen central memory T cells (TCM, CD 3) were analyzed by flow cytometry⁺CD8⁺CD62L⁺CD44⁺) And effector memory T cells (TEM, CD 3)⁺CD8⁺CD62L^-CD44⁺) As shown in fig. 5a, it was shown that there was no difference in the proportion of central memory T cells in the Imiquimod + OX40 agonist antibody group compared to the NS treated group, while the proportion of effector memory T cells increased by 1-fold, suggesting that TEM is able to induce strong immunoprotection by producing TNF- α and IFN- γ. Mouse splenocytes from the NS and Imiquimod + OX40 agonist antibody groups were compared with CFSE-labeled H₂₂Hepatoma cells were incubated at effector-to-target ratios (E: T) of 5:1, 10:1 and 20: 1. After 6H incubation, PI was added and the tumor cells were analyzed by flow cytometry for the proportion of dead cells (CFSE + PI +), as shown in FIG. 5b, which shows that splenocytes from mice in the Imiquimod + OX40 agonist antibody group were directed against H at an effective target ratio of 10:1₂₂The liver cancer cell shows more obvious killing capability. One week after completion of the last treatment, tumor microenvironment was analyzed by flow cytometry for macrophages of type M2 (CD11 b)⁺F4/80⁺CD206⁺) The results are shown in figure 5c, showing that the combined intratumoral injection of imiquimod and OX40 agonist antibodies results in a reduction of M2-type macrophages, and the level of the cytokine IL-10 secreted mainly therefrom.

The immune response triggered by the intratumoral injection of the compound immunopotentiator has specificity. As shown in fig. 6a, the treatment regimen was: kunming mice with complete tumor regression after intratumoral injection of the compound immunopotentiator are subcutaneously inoculated with B16F10 melanoma cells (2X 10) again 40 days after the first tumor inoculation⁵Left lower abdomen) And H₂₂Liver cancer cell (2X 10)⁶Right lower abdomen). The tumor growth curve for each mouse (5 mice) is shown in figure 6 b. The results show that mice cured by in situ vaccination are against the same tumor (H)₂₂Hepatocellular carcinoma, right lower abdomen) had strong immune resistance, and the liver cancer foci did not recur. While a different tumor (B16F10 melanoma, left lower abdomen) formed a relatively large tumor lesion subcutaneously in mice.

Claims

1. A composite immunopotentiator for tumor therapy, which is characterized by:

comprises imiquimod suspension and OX40 solution;

imiquimod suspensions include imiquimod;

solutions of OX40 include OX40 agonist antibodies.

2. The composite immunopotentiator according to claim 1, wherein:

the auxiliary material is sodium caprylate solution.

3. The composite immunopotentiator according to claim 1, wherein:

wherein the imiquimod suspension also comprises a Tween 80 solution and Montanide ISA 51; the Tween 80 solution is prepared by dissolving Tween 80 in normal saline and pure water.

4. The composite immunopotentiator according to claim 1, wherein:

wherein the OX40 solution is prepared by dissolving OX40 agonist antibody in physiological saline.

5. The method for producing a composite immunopotentiator according to any one of claims 1 to 4, wherein:

the method comprises the following steps:

6. The method for preparing a composite immunopotentiator according to claim 5, wherein:

wherein the concentration of the physiological saline is 0.9 m/v%; in the Tween 80 solution, the volume ratio of normal saline, pure water and Tween 80 is 94.4: 5.4: 0.2;

in the imiquimod suspension, the dosage ratio of the Tween 80 solution to the Montanide ISA51 is 1ml to 40 mu l, and the dosage ratio of the mixed emulsion to the imiquimod is 1ml to 2 mg.

7. The method for preparing a composite immunopotentiator according to claim 5, wherein:

wherein, in the OX40 solution, the dosage ratio of the OX40 agonist antibody to the physiological saline is 10 mg: 1 ml.

8. The method for preparing a composite immunopotentiator according to claim 5, wherein:

wherein, in the sodium caprylate solution, the dosage ratio of the sodium caprylate to the normal saline is 15 mg: 1.5 ml.

9. The method for preparing a composite immunopotentiator according to claim 6, wherein:

wherein the volume ratio of the imiquimod suspension, the OX40 solution and the sodium caprylate solution is 1.5: 1: 1.5.

10. Use of the composite immunopotentiator according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of tumors.