IMMUNE AGONIST COMPLEX, AND PREPARATION AND APPLICATION THEREOF

Abstract

A natural immune agonist complex, consisting of an immune agonist and a targeted liposome, where the immune agonist is M(cGAMP)L.sub.n. The targeted liposome is formed by a nanobody targeting a tumor microenvironment, a cell membrane-targeted penetrating peptide, or a blood-brain barrier-targeted penetrating peptide with a liposome through chemical bonding. This application further provides a preparation and application of the natural immune agonist complex.

Claims

1. An immune agonist complex, wherein the immune agonist complex consists of a natural immune agonist and a targeted liposome; the targeted liposome is formed by a nanobody targeting a tumor microenvironment, a cell membrane-targeted penetrating peptide, or a blood-brain barrier-targeted penetrating peptide with a liposome through chemical bonding; and the natural immune agonist is M(cGAMP)L.sub.n, wherein M is an ion of a transition metal; L is a ligand containing 0, N or S, and n is selected from 0-2.

2. The immune agonist complex of claim 1, wherein the transition metal is selected from the group consisting of zinc, manganese, copper, and ruthenium; the nanobody is an anti-programmer death-1 (anti-PD-1) nanobody, an anti-programmed death ligand-1 (anti-PDL-1) nanobody, an anti-CD-47 nanobody, or an anti-transferrin receptor 1 (anti-TfR1) nanobody; the ligand is selected from the group consisting of 5-fluorouracil, imidazole, gemcitabine, capecitabine, water, and 6-mercaptopurine; the cell membrane-targeted penetrating peptide is gH625 consisting of SEQ ID NO: 1; and the blood-brain barrier-targeted penetrating peptide is PT8 consisting of SEQ ID NO: 2.

3. The immune agonist complex of claim 2, wherein the immune agonist complex is selected from the group consisting of: MncGAMP-anti-human PD-1 nanobody-liposome (complex I); MncGAMP-anti-human PD-L1 nanobody-liposome (complex II); MncGAMP-anti-human CD47 nanobody-liposome (complex III); MncGAMP-anti-mouse PD-1 nanobody-liposome (complex IV); MncGAMP-anti-mouse PD-L1 nanobody-liposome (complex V); MncGAMP-anti-mouse CD47 nanobody-liposome (complex VI); ZncGAMP-gH625-liposome (complex VII); ZncGAMP-anti-mouse TfR1 nanobody-liposome (complex IX); and ZncGAMP-gH625-PT8-liposome (complex X).

4. A method of preparing the immune agonist complex of claim 1, comprising: preparing the natural immune agonist M(cGAMP)L.sub.n; preparing the nanobody; subjecting the nanobody to terminal thiolation to obtain a terminally-thiolated nanobody; preparing an unilamellar liposome; and adding the natural immune agonist M(cGAMP)L.sub.n and the terminally-thiolated nanobody to the unilamellar liposome in sequence followed by incubation to obtain the immune agonist complex.

5. The method of claim 4, wherein the natural immune agonist M(cGAMP)L.sub.n is prepared through steps of: reacting cGAMP with a transition metal salt under stirring and heating in the presence of the ligand followed by purification using an ion-exchange column to obtain the natural immune agonist M(cGAMP)L.sub.n.

6. The method of claim 4, wherein the nanobody is prepared by using an Escherichia coli (E. coli) expression system.

7. The method of claim 4, wherein the terminal thiolation is performed through steps of: adding ethylene diamine tetraacetic acid (EDTA) to a solution of the nanobody to obtain a mixture, wherein a concentration of the EDTA in the mixture is 5 mM; adding a thiolation reagent dropwise into the mixture followed by incubation in the dark for 1 h, wherein the thiolation reagent is Traut's reagent; and removing excess thiolation reagent by using a de-salting column to obtain the terminally-thiolated nanobody.

8. The method of claim 4, wherein the unilamellar liposome is prepared through steps of: dissolving lecithin, cholesterol, and 1,2-distearoyl-SN-glycerol-3-phosphorylethanolamine-N-maleimide-polyethylene glycol in chloroform followed by vacuum rotary evaporation in a water bath and addition of (NH.sub.4).sub.2SO.sub.4 to obtain the unilamellar liposome.

9. A method for treating a tumor in a subject in need thereof, comprising: administering a therapeutically effective amount of the immune agonist complex of claim 1 to the subject; wherein the tumor is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, testicular cancer, lung cancer, nasopharyngeal carcinoma, esophageal cancer, kidney cancer, glioma, melanoma, malignant lymphoma, head and neck cancer, thyroid cancer, and osteosarcoma; and an administration route is selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intravenous drip, intranasal drip, oral administration, and a combination thereof.

10. A method for treating viral infection in a subject in need thereof, comprising: administering a therapeutically effective amount of the immune agonist complex of claim 1 to the subject; wherein the viral infection is caused by coronavirus, an influenza virus, or human immunodeficiency virus (HIV); and an administration route is selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intravenous drip, intranasal drip, oral administration, and a combination thereof.

11. A method for treating viral inflammation in a subject in need thereof, comprising: administering a therapeutically effective amount of the immune agonist complex of claim 1 to the subject; wherein the viral inflammation is Corona Virus Disease 2019 (COVID-19), viral nephritis, viral encephalitis, viral enteritis, or viral hepatitis; and an administration route is selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intravenous drip, intranasal drip, oral administration, and a combination thereof.

12. A method for treating tumor metastasis in a subject in need thereof, comprising: administering a therapeutically effective amount of the immune agonist complex of claim 1 to the subject; wherein the tumor metastasis is lung metastasis, liver metastasis, lymphatic metastasis, or brain metastasis; and an administration route is selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intravenous drip, intranasal drip, oral administration, and a combination thereof.

13. A method for treating a neurodegenerative disease in a subject in need thereof, comprising: administering a therapeutically effective amount of the immune agonist complex of claim 1 to the subject; wherein the neurodegenerative disease is Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis, ataxia telangiectasia (AT), bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease (CJD), Huntington's disease, spinocerebellar atrophy, spinal muscular atrophy, spastic paraplegia, or myasthenia gravis; and an administration route is selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intravenous drip, intranasal drip, oral administration, and a combination thereof.

14. A method for treating a brain disease in a subject in need thereof, comprising: administering a therapeutically effective amount of the immune agonist complex of claim 1 to the subject; wherein the brain disease is ischemic cerebrovascular injury, craniocerebral injury, encephalitis, or brain tumor; and an administration route is selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intravenous drip, intranasal drip, oral administration, and a combination thereof.

15. A pharmaceutical composition, comprising: the immune agonist complex of claim 1; and a pharmaceutically acceptable excipient; wherein the pharmaceutical composition is in a form of injection, drop, or oral preparation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0075] FIG. 1a is a mass spectrum of a natural immune agonist MncGAMP according to an embodiment of the present disclosure;

[0076] FIG. 1b is a mass spectrum of a natural immune agonist ZncGAMP according to an embodiment of the present disclosure;

[0077] FIG. 2a is a nuclear magnetic resonance (NMR) spectrum of the natural immune agonist MncGAMP according to an embodiment of the present disclosure;

[0078] FIG. 2b is an NMR spectrum of the natural immune agonist ZncGAMP according to an embodiment of the present disclosure; and

[0079] FIG. 3 schematically illustrates inhibition effects of a novel immune agonist complex prepared herein on pneumonia in mice (H&E staining of lung tissue paraffin sections).

DETAILED DESCRIPTION OF EMBODIMENTS

[0080] The present application will be clearly and completely described below with reference to the embodiments. Obviously, described below are intended to better illustrate the present application, rather than limit the scope of the present application.

Example 1 Preparation of a Novel Immune Agonist Complex

[0081] (S1) Synthesis of Immune Agonist and Metal Complex Thereof

[0082] The cyclic dinucleotide cGAMP was synthesized under the catalysis of a cyclic GMP-AMP synthetase (cGAS), and had a purity of above 98%. An immune agonist metal complex ([M(cGAMP)L.sub.0.2]) was prepared through reaction of a transition metal salt and the immune agonist (i.e., cGAMP) under stirring and heating in the presence of the small-molecular ligand followed by purification by using an ion exchange column, where M was a transition metal ion, i.e., Zn, Mn, Cu, or Ru; and L was a small-molecular ligand containing 0, N or S, such as 5-fluorouracil, imidazole, gemcitabine, capecitabine, water, and 6-mercaptopurine. The immune agonist metal complex was analyzed for the metal content and element composition.

[0083] FIG. 1a and 1b were respectively mass spectra of MncGAMP and ZncGAMP, where the peak at m/z 673.09 corresponded to the peak of cGAMP anion, shown as:

##STR00001##

[0084] NMR spectra of MncGAMP and ZncGAMP were respectively shown in FIG. 2a and 2b, characterized as:

[0085] MncGAMP: .sup.lH NMR (400 MHz, D.sub.2O) δ 8.27 (s, 1H), 7.93 (s, 1H), 7.82 (s, 1H), 5.85 (s, 1H), 5.71 (s, 1H), 5.51 (s, 1H), 4.31 (d, J=21.3 Hz, 3H), 4.17 (s, 3H), 3.96 (s, 1H); and

[0086] ZncGAMP: .sup.lH NMR (400 MHz, D.sub.2O) δ 8.22 (s, 1H), 8.03 (s, 1H), 7.69 (s, 1H), 5.92 (s, 1H), 5.79 (d, J=7.7 Hz, 1H), 5.59 (s, 1H), 4.82 (s, 2H), 4.44 (d, J=21.7 Hz, 2H), 4.22 (d, J=49.6 Hz, 5H), 3.87 (s, 1H).

[0087] (S2) Preparation of a Nanobody

[0088] Nanobodies (e.g., anti-PD1, anti-PDL1, anti-CD47, and anti-TfR1 nanobodies) were human (mouse) monoclonal nanobodies, where the plasmid adopted PET-22 B (+) as a carrier, carrying AMP+resistance, and a terminal of the protein sequence was marked with 6 His-Tag to facilitate the purification. The nanobodies were efficiently expressed by Escherichia coli, and purified by an affinity column with a purity of 98%. The freeze-dried powder was stored in an ultra-low temperature refrigerator for later use. The targeted penetrating peptides were synthesized by a biotechnological company.

[0089] (S3) Preparation of Nanobody/Penetrating Peptide-Liposome-Immune Agonist Complexes

[0090] A nanobody/penetrating peptide was subjected to terminal thiolation. After that, a solution of the nanobody was added with ethylene diamine tetraacetic acid (EDTA) to form a mixture, where a concentration of the EDTA in the mixture was 5 mM. The mixture was then added with a thiolation reagent (Traut's reagent) under stirring. The thiol groups on the nanobody were determined by Ellman's method to verify the successful thiolation of the nanobody. Liposomal materials (including lecithin, cholesterol, and 1,2-distearoyl-SN-glycerol-3-phosphorylethanolamine-N-maleimide-polyethylene glycol 2000) were dissolved in chloroform and dried into a film by vacuum rotary evaporation in a water bath followed by addition of (NH.sub.4).sub.2SO.sub.4 for hydration to obtain a unilamellar liposome. A blank liposome was added with an immune agonist/immune agonist metal complex and the terminally-thiolated nanobody followed by incubation in the dark overnight and removal of unencapsulated drug and unattached nanobody proteins by a molecular sieve column to obtain the immune agonist complex. The immune agonist complex was examined by transmission electron microscope (TEM), which had bilayer round vesicles, good morphology, liposome diameter of about 200 nm, and zeta potential of about 24 mV. The immune agonist complex had an immune agonist encapsulation rate of 80%, and was stable under refrigeration at 4° C. The lyophilized powder of 3% trehalose solution was stored under refrigeration. The novel immune agonist complexes prepared in Example 1 were as follows:

[0091] MncGAMP-anti-human PD-1 nanobody-liposome (complex I);

[0092] MncGAMP-anti-human PD-L1 nanobody-liposome (complex II);

[0093] MncGAMP-anti-human CD47 nanobody-liposome (complex III);

[0094] MncGAMP-anti-mouse PD-1 nanobody-liposome (complex IV);

[0095] MncGAMP-anti-mouse PD-L1 nanobody-liposome (complex V);

[0096] MncGAMP-anti-mouse CD47 nanobody-liposome (complex VI);

[0097] ZncGAMP-gH625-liposome (complex VII);

[0098] McGAMP-liposome (M=Mn/Zn) (complex VIII)

[0099] ZncGAMP-anti-mouse TfR1 nanobody-liposome (complex IX); and

[0100] ZncGAMP-gH625-PT8-liposome (complex X).

Example 2 Evaluation of Antitumor Effects of Novel Immune Agonist Complexes

Experimental Animals

[0101] Specific pathogen free (SPF)-grade BALB/C and C.sub.57BL/6 male mice, aged 7-8 weeks and weighing 20-22 g, were purchased from Shanghai Slac Laboratory Animal Co., Ltd (Laboratory animal quality certificate number: SCXK (Shanghai)2007-0005.

Feeding Conditions

[0102] All mice were fed freely with sterilized food and water, and kept at room temperature (23±2° C.), where the entire feeding process met the requirements of SPF grade.

Dose Design

[0103] Mice were injected intraperitoneally with the immune agonist or complex with a dose of 10 mg/kg, or each injected intraperitoneally with 200 μg of the nanobody.

Control Test

[0104] Negative control: PBS solution.

[0105] Positive control: cGAMP with a dose of 10 mg/kg.

Administration

[0106] Administration route: intraperitoneal injection.

[0107] Dose of the novel immune agonist complex: 100 μL/each mouse.

[0108] Dose of the nanobody (anti-PD-L1, anti-CD47, and anti-TfR1 nanobody): 200 μg/each mouse.

[0109] Frequency: once a day for consecutive 21 days.

[0110] The number of mice in each group: 10.

Cell Material

[0111] Murine colorectal cancer cell line CT26, murine breast cancer cell line 4T1, and murine lung cancer cell line LL/2 were all purchased from the Cell Bank of the Chinese Academy of Sciences.

Experimental Procedures

Establishment and Intervention of Tumor Model Mice

[0112] Cancer cells were cultured, passaged, collected at the logarithmic phase of cells, and prepared into a cell suspension at a concentration of (1.0×10.sup.7) per ml. Mice were injected with 0.2 ml of the cell suspension at the axilla of the right forelimb (cell number 2.0×106 cells/each), and were tumorigenic in about 8 days. These mice were randomly divided into 10 groups equally, namely, group A: negative control group (saline group), group B: positive control (cGAMP) group (dose: 10 mg/kg), group C: MncGAMP group (dose: 10 mg/kg), group D: anti-PD-L1 nanobody group, group E: anti-CD47 nanobody group, group F: anti-TfR1 nanobody group, group G: complex V group (dose: 10 mg/kg), group H: complex VI group (dose: 10 mg/kg), group I: complex IX group (dose: 10 mg/kg), and group J: complex VIII group (10 mg/kg). The mice were administered once a day for consecutive 21 days. 21 days later, the mice were executed and the tumor weights were weighed, and the tumor inhibition rate was calculated by: [1−average tumor weight of experimental groups (groups B, C, D, E, F, G, H, I, and J)/average tumor weight of group A]]×100%.

[0113] Murine colorectal cancer cell line CT26 was prepared and transplanted into the BA1B/C mice, murine breast cancer cell line 4T1 was prepared and transplanted into BA1B/C mice, and murine lung cancer Lewis tumor line LL/2 was prepared and transplanted into C.sub.57BL/6 mice to evaluate the anti-tumor effects of different drugs.

Statistical Analysis

[0114] The data were expressed as ×±s and processed by SPSS10.0 software. One-way ANOVA test was used to compare the significance of the difference in tumor weight among the groups, and the significance level (a) was 0.05.

Experimental Results

[0115] A subcutaneous transplantation tumor model was prepared by subcutaneous inoculation of tumor cells in mice. The novel immune agonist complexes prepared herein all significantly inhibited the growth of the tumor, and the tumor weights were significantly lower than those of the negative control group (P<0.05, P<0.01) after 21 days of the administration, indicating that the immune agonist complexes had greatly improved antitumor effects. The specific results were shown in Table 1.

TABLE-US-00001 TABLE 1 Inhibition effect of novel immune agonist complexes on BALB/C mice transplanted with colorectal cancer cell line CT26 (n = 10, mean ± SD) Average tumor Average tumor Groups weight (g) inhibition rate (%) Negative control group 2.244 ± 0.266 — Positive control (cGAMP) 0.784 ± 0.135 65.0 group MncGAMP group 0.493 ± 0.203 78.0 MncGAMP-Lipo 0.404 ± 0.187 81.9 (complex VIII) group Anti-PD-L1 nanobody 1.773 ± 0.185 22.7 group Anti-CD47 nanobody 1.907 ± 0.205 15.0 group Anti-TfR1 nanobody 1.819 ± 0.195 18.9 group Complex V group 0.165 ± 0.103 92.6 Complex VI group 0.218 ± 0.116 90.3 Complex IX group 0.309 ± 0.128 86.2 Noted: *P < 0.05 vs negative control group; and **P < 0.01 vs negative control group.

TABLE-US-00002 TABLE 2 Inhibition effect of novel immune agonist complexes on C57BL/6 mice transplanted with lung cancer Lewis tumor line LL/2 (n = 10, mean ± SD) Average tumor Average tumor Groups weight (g) inhibition rate (%) Negative control group 2.846 ± 0.208 — Positive control (cGAMP) 1.138 ± 0.127 60.0 group MncGAMP group 0.578 ± 0.212 79.7 Complex VIII group 0.512 ± 0.207 82.0 Anti-PD-L1 nanobody 2.217 ± 0.156 22.1 group Anti-CD47 nanobody 1.995 ± 0.212 29.9 group Anti-TfR1 nanobody 2.277 ± 0.168 19.9 group Complex V group 0.316 ± 0.206 88.9 Complex VI group 0.285 ± 0.163 90.0 Complex IX group 0.365 ± 0.124 87.2 Noted: *P < 0.05 vs negative control group; and **P < 0.01 vs negative control group.

TABLE-US-00003 TABLE 3 Inhibition effect of novel immune agonist complexes on BALB/C mice transplanted with breast cancer cell line 4T1 (n = 10, mean ± SD) Average tumor Average tumor Groups weight (g) inhibition rate (%) Negative control group 2.268 ± 0.282 — Positive control (cGAMP) 0.862 ± 0.156 61.9 group MncGAMP group 0.463 ± 0.197 79.6 Complex VIII group 0.384 ± 0.201 83.1 Anti-PD-L1 nanobody 1.856 ± 0.215 18.2 group Anti-CD47 nanobody 1.808 ± 0.208 20.3 group Anti-TfR1 nanobody 1.915 ± 0.184 15.6 group Complex V group 0.286 ± 0.162 87.4 Complex VI group 0.208 ± 0.186 90.8 Complex IX group 0.317 ± 0.201 86.0 Noted: *P < 0.05 vs negative control group; and **P < 0.01 vs negative control group.

Example 3 Evaluation of Metastasis of Murine Breast Cancer 4T1-Luc of Novel Immune Agonist Complexes

Experimental Animals

[0116] Specific pathogen free (SPF)-grade BALB/C male mice, aged 7-8 weeks and weighing 20-22 g, were purchased from Shanghai Slac Laboratory Animal Co., Ltd (Laboratory animal quality certificate number: SCXK (Shanghai)2007-0005.

Feeding Conditions

[0117] All mice were fed freely with sterilized food and water, and kept at room temperature (23±2° C.), where the entire feeding process met the requirements of SPF grade.

Dose Design

[0118] Mice were injected intraperitoneally with the immune agonist complex or MncGAMP with a dose of 10 mg/kg, or each injected intraperitoneally with 200 μg of the nanobody.

Control Test

[0119] Negative control: PBS solution.

[0120] Positive control: cGAMP with a dose of 10 mg/kg.

Administration

[0121] Administration route: intraperitoneal injection.

[0122] Dose of the novel immune agonist complex or MncGAMP: 100 μL/each mouse.

[0123] Dose of the nanobody (anti-PD-L1 and anti-CD47 nanobodies): 200 μg/each mouse.

[0124] Frequency: once a day for consecutive 21 days.

[0125] The number of mice in each group: 10.

Cell Material

[0126] Used herein was murine breast cancer cell line 4T1-luc (luciferase labeled tumor cells), which was provided by medical department of Zhejiang University School. The 4T1-luc cell line in BALB/C mice had similar growth and metastatic properties to breast tumors in humans, which was an animal model for VI-stage breast cancer in humans. The 4T1-luc cell line spontaneously produced highly metastatic tumors that metastasize to the lung, liver, lymph nodes, and brain, while forming the primary site at the injection site. Luciferase was a general term for a class of enzymes in living organisms that catalyze the oxidative luminescence of luciferin or firefly aldehyde, which was derived from organisms naturally capable of luminescence.

Experimental Procedures

[0127] (1) Establishment and intervention of tumor model mice.

[0128] Cancer cells were cultured, passaged, collected at the logarithmic phase of cells, and prepared into a cell suspension at a concentration of (1.0×10.sup.7) per ml. Mice were injected with 0.2 ml of the cell suspension at the axilla of the right forelimb (2.0×10.sup.6 cells/each), and were injected with drugs the day after inoculation with breast cancer cells. These mice were randomly divided into 9 groups equally, namely, group A: negative control group (saline group), group B: positive control (cGAMP) group (dose: 10 mg/kg), group C: complex VIII group (dose: 10 mg/kg), group D: anti-PD-L1 nanobody group, group E: anti-CD47 nanobody group, group F: anti-TfR1 nanobody group, group G: complex V group (dose: 10 mg/kg), group H: complex VI group (dose: 10 mg/kg), group I: complex IX group (dose: 10 mg/kg). The mice were administered once a day for consecutive 30 days.

[0129] (2) Detection of Effects of Different Drugs on Anti-Metastasis of Breast Cancer

[0130] Metastasis of murine breast cancer in mice was detected using a small animal in vivo optical imaging system (Perkin Elmer, IVIS Lumina XRMS Series III) on days 5, 10, 20, and 30 after drug administration, respectively, which was done at Shanghai Medical College of Fudan University. The effects of different drugs on anti-metastasis of murine breast cancer were summarized in Table 4.

TABLE-US-00004 TABLE 4 Effects of different drugs on anti-tumor metastasis of breast cancer Groups Day 5 Day 10 Day 20 Day 30 Negative control No Lung Lung, liver, Lung, liver, group and hindquarter, hindquarter and brain Positive control No No No Hindquarter (cGAMP) group and hind leg Complex VIII No No No hindquarter group Anti-PD-L1 No No Lung and Lung, nanobody group hindquarter hindquarter, and brain Anti-CD47 No No Lung and Lung, nanobody group hindquarter hindquarter, and hind leg Anti-TfR1 No No Lung and Lung and nanobody group hindquarter hindquarter Complex V group No No No No Complex VI group No No No No Complex IX group No No No No

[0131] The experimental results showed that the novel immune agonist complexes were significantly superior to the immune agonists and metal complexes thereof alone, and even better than the nanobody (anti-PD-L1 or anti-CD47) alone, in anti-metastasis of murine breast cancer 4T1-Luc on BALB/C mice. Therefore, the novel immune agonist complexes prepared herein had potential clinical application against tumor metastasis.

Example 4 Inhibition Effects of Novel Immune Agonist Complexes on Coronavirus Replication

[0132] Peripheral blood mononuclear cells (PBMCs) used herein was purchased from Shanghai Saili biotechnology Co., Ltd, which mainly included lymphocytes (T cells/B cells), monocytes, macrophages, and dendritic cells. Most of PBMCs were lymphocytes. PBMCs were normal primary cells, which belonged to a mixed system.

[0133] Cells freezing conditions: 90% complete medium was added with 10% DMSO, and stored in liquid nitrogen.

[0134] Quality control (QC) tests confirmed that the culture system was free of HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi.

[0135] The virus strain used herein was coronavirus (ATCC VR-841), which was suitable for laboratory use and purchased from ATCC company. It was a bronchopneumonia coronavirus. Virus experiments in this study were performed by virus laboratory of American Animals Inc.

[0136] Culture operations of PBMCs were described below.

[0137] (1) Cell Thawing

[0138] The frozen tube containing 1 mL of cell suspension was thawed by shaking rapidly in a 37° C. water bath, added with 4 mL of culture medium followed by mixing and centrifugation at 1000 RPM for 4 min. Then, the supernatant was discard, and 1-2 mL of medium was added followed by blowing evenly. After that the cell suspension was added to the culture flask and cultured overnight. The cell suspension was changed and cell density was checked in the next day.

[0139] (2) Cell Passage

[0140] When the cell density reached 80%-90%, the passage culture was performed. The culture supernatant was discarded, and the cells was rinsed with PBS solution for 1-2 times, where the solution did not contain calcium and magnesium ions. 1 ml of digestive juice (0.25% of Trypsinolide-0.53 Mary EDTA) was added into a culture bottle, digested in a 37° C. incubator for 1-2 min, and then observed under a microscope. If most of the cells became rounded and fell off, the culture bottle was taken back to the operation table quickly, tapped and then added with a small amount of culture medium to end the digestion. Each culture bottle was added with 8 mL of medium, and tapped followed by sucking the medium out. Then the culture bottle was centrifuged at 1000 rpm for 4 min, the supernatant was discarded, and 1-2 mL of culture solution was added into the culture bottle followed by blowing uniformly. The cell suspension was divided and respectively added into two new bottles containing 8 ml of culture medium according to a ratio of 1:2. Cell viability was determined by a fixable red dead cell stain kit (Life Technology Company). Cells were stained with 0.5 μL of dye in 1 mL of PBS solution in the dark for 5 min, and then washed twice with PBS solution and measured on FACS Calibur. As a positive control of dead cells, PBMCs were boiled at 95° C. for 20 min and stained with the same procedure to determine the peaks of dead cells.

[0141] The cultured PBMCs were divided into seven groups (six samples in each group), namely, group A: negative control group (PBMC); group B, PBMC+cGAMP; group C, PBMC+MncGAMP; group D, PBMC+complex VIII; group E, PBMC+complex I; group F, PBMC+complex II; and group G, PBMC+complex III. Except for group A, which was used as a control group without drug addition, all other groups were respectively added with 100 μg/mL of the corresponding drug. Each cell sample was added with 10 μL of virus, and the antiviral status of the different immune agonist complexes were determined after three weeks. The inhibition rate of virus was selected as an evaluation index. The activity titer of the virus was detected by fluorescence quantitative RT-PCR. The effects of various immune agonist complexes on viral inhibition were listed in Table 5.

TABLE-US-00005 TABLE 5 The inhibition rate of coronavirus by immune agonist complexes Average inhibition rate of Groups coronavirus (%) A — B 25 ± 4 C 49 ± 7 D 62 ± 3 E 92 ± 5 F 90 ± 6 G 87 ± 5

[0142] Table 5 showed that the novel immune agonist complexes all had good inhibitory effects on the replication of coronavirus in PBMC cells, superior to a bare drug cGAMP or cGAMP-non-targeted liposome (complex VIII). Moreover, the immune agonist metal complex MncGAMP had a significantly improved inhibitory effect compared with a negative electronegative bare cGAMP. Therefore, the novel immune agonist complexes showed remarkably improved virus inhibition effects.

Example 5 Inhibition Effects of Novel Immune Agonist Complexes on Viral Pneumonia in Mice

Experimental Animals.

[0143] Specific pathogen free (SPF)-grade C.sub.57BL/6 male mice, aged 7-8 weeks and weighing 20-22 g, were purchased from American Animals Inc.

Feeding Conditions

[0144] All mice were fed freely with sterilized food and water, and kept at room temperature (23±2° C.), where the entire feeding process met the requirements of SPF grade.

Animal Grouping

[0145] 42 mice were randomly divided into seven groups (six mice in each group), namely, group A, normal control group; group B, pneumonia model group, PBS; group C, administration with MncGAMP (dose: 10 mg/kg); group D, administration with complex VIII (dose: 10 mg/kg); group E, administration with complex IV (dose: 10 mg/kg); group F, administration with complex V (dose: 10 mg/kg); and group G, administration with complex VI (dose: 10 mg/kg).

Establishment of Pneumonia Virus Model Mice

[0146] Mice were placed under sufficiently deep anesthesia, secured in a dorsal recumbent position, and dropped with VR-841 virus suspension slowly through the inner walls of the nostrils in a volume of 60 μL (30 μL in per nostril) to ensure maximum efficiency of pulmonary infection. After that, the mice were gently removed from the bench, and the head and chest were padded with folded paper towels in small increments to ensure smooth breathing. After the mice were awakened, they were placed back into the mouse cage.

Lung Histopathological Observation

[0147] The tissue of the left lung lobes was taken, cut in half, fixed with picric acid solution for 24 hours, dehydrated, transparent, embedded by paraffin, sectioned in a thickness of 5 and then stained with hematoxylin-eosin (HE). The pathomorphological changes of the lung tissue were observed under light microscopy.

Preparation of Mouse Alveolar Lavage Solution.

[0148] An equal volume of PBS was taken and injected along the trachea of mice and then sucked out, so a few times, the alveolar lavage fluid was obtained. An equal volume of DMEM was injected intraperitoneally into the blank group and the pneumonia model group. Collected serum was stored at −80° C. The ELISA method was used to detect concentrations of TNF-alpha and IL-1beta according to the kit instructions. After the reaction was terminated, the enzyme plate was placed into the enzyme standardizer slot for detection with a 450 nm wavelength. The standard and blank control areas were identified, the corresponding optical density values were detected, and then the standard curve was plotted and the corresponding concentrations were calculated. In the mouse pneumonia model, the levels of pro-inflammatory cytokines IL-1beta and TNF-alpha were significantly increased in both serum and alveolar lavage fluid, and both were reduced to varying degrees by the administration of immune agonist complexes. The effects of the different drugs on inhibition of pneumonia in mice were shown in Table 6.

[0149] The experimental results showed that the novel immune agonist complexes IV/V/VI showed better effects on anti-inflammatory cytokines in mouse than the bare immune agonist, immune agonist metal complex, and simple immune agonist liposome. Therefore, the novel immune agonist complexes had effects on anti-inflammatory damage in mice.

TABLE-US-00006 TABLE 6 Inhibition effects of novel immune agonist complexes on viral pneumonia in mice Serum (pg/mL) Alveolar lavage fluid (pg/mL) Groups TNF-α IL1β TNF-α IL1β A  500 ± 35  50 ± 12 120 ± 43 10 ± 5  B 1500 ± 78 550 ± 35 500 ± 56 70 ± 16 C  960 ± 56 120 ± 15 310 ± 78 45 ± 14 D  850 ± 75  90 ± 18 220 ± 32 30 ± 18 E  610 ± 37  70 ± 12 160 ± 43 22 ± 15 F  620 ± 48  65 ± 14 150 ± 26 25 ± 11 G  580 ± 86  76 ± 16 180 ± 46 18 ± 8 

[0150] As shown in FIG. 3, compared with the normal group, the mice in the pneumonia model group had an increased infiltration of inflammatory cells in the lungs and a significantly increased thickness of the alveolar septum. After the administration of the immune agonist MncGAMP, the symptoms of lung inflammation in mice were alleviated. Unluckily, the bare MncGAMP immune agonist was less effective, while the novel immune agonist complexes showed significantly improved efficacy.

Example 6 Evaluation of Immune Adjuvant Function of Novel Immune Agonist Complexes

Experimental Animals

[0151] Specific pathogen free (SPF)-grade C.sub.57BL/6 male mice, aged 7-8 weeks and weighing 20-22 g, were purchased from American Animals Inc. The aluminum adjuvant and OVA were purchased from Invitrogen company.

Mice Grouping

[0152] The mice were divided into 7 groups (10 mice in each group), namely, group A: OVA+cGAMP; group B: OVA+MncGAMP; group C: OVA+complex VIII; group D: OVA+complex IV; group E: OVA+complex VII; group F: OVA+aluminum adjuvant; and group G: OVA.

[0153] Each mouse was injected subcutaneously with 10 μg of OVA and 100 μg of aluminum adjuvant or a different species of cGAMP (or the immune agonist complex), and those not injected were negative control groups. Immunization was performed once on days 1, 7, and 14, respectively, and lung lavage fluid was obtained and blood samples taken on day 21. The potency of the immune agonist and the complex as adjuvants to induce nanobody production was determined by ELISA, and the results were shown in Table 7. The results showed that both the immune agonist MncGAMP and immune agonist complex significantly induced immune nanobodies, superior to the aluminum adjuvant, and the novel immune agonist complexes also had higher effects in contrast to the naked cGAMP and MncGAMP.

TABLE-US-00007 TABLE 7 Immune adjuvant effects of novel immune agonist complexes Alveolar lavage fluid Groups Serum (IgG) (IgA) A 17.5 ± 0.9 2.3 ± 0.2 B 19.2 ± 1.2 3.5 ± 0.8 C 25.6 ± 1.5 4.2 ± 0.7 D 31.8 ± 1.8 6.5 ± 0.6 E 28.4 ± 2.1 5.6 ± 1.1 F 21.5 ± 1.6 2.8 ± 0.5 G 10.6 ± 1.2 1.1 ± 0.2 Noted: Both IgG and IgA are values of Iog2.

Example 7 Effect of Novel Immune Agonist Complexes on Activation of Immune Cells

[0154] Mice were raised, immunized with virus infection, and subjected to blood collection, as the same as Example 3. Isotype control nanobody was purchased from eBiosciences; antibody magnetic beads were purchased from Militeny Biotech; and flow cytometer was purchased from BD company. 14 days after immunization, mouse spleen and lung tissues were taken, ground and pounded separately, filtered with a 40-μm filter, and centrifuged at 1000 rpm for 10 min to separate unlysed immune cells. The DC cells (CD40\CD80\CD86\MHCII), T cells and (CD8+) cells were separated by the antibody magnetic beads, and added with the corresponding FAC nanobody (diluted with FACS buffer). As a negative control, the isotype control nanobody was added into the DC cells followed by incubation for 1 h, centrifugation, and rinsing with PBS. After that, the samples were analyzed by the flow cytometry to select the appropriate cells for determination of fluorescence intensity (MFI), and the results were shown in Table 8. The results showed that both the immune agonist MncGAMP and immune agonist-targeted liposome complexes significantly activated dendritic cell DCs and T cells, and the effect of the novel immune agonist complexes was significantly higher in contrast to the naked drugs cGAMP and MncGAMP.

TABLE-US-00008 TABLE 8 Effect of novel immune agonist complexes on induced activation of immune cells CD40 CD80 CD86 MHCII CD8 T cells cells cells cells cells cells Groups (%) (%) (%) (%) (%) (%) A (cGAMP) 122.6 115.5 18.3 218.4 2.3 ± 0.2 0.3 ± 0.2 B 155.8 123.6 21.4 266.5 3.5 ± 0.8 0.5 ± 0.3 (MncGAMP) C (complex 186.2 156.6 19.7 250.8 4.2 ± 0.7 0.7 ± 0.2 VIII) D (complex 256.4 238.2 36.7 342.8 8.9 ± 0.6 1.3 ± 0.1 V) E (complex 284.8 243.6 42.5 421.2 9.2 ± 1.1 1.1 ± 0.3 VII) F (blank) 36.9 15.3 5.2 120.8 0.8 ± 0.5 0.2 ± 0.3 G (isotype 5.3 3.3 1.2 8 0.6 ± 0.2 0.1 ± 0.1 control nanobody)

Example 8 Confirmation of Effects of Novel Immune Agonist Complexes on Cognitive Ability of AD Mice by Morris Water Maze Test

[0155] APP/PS1 transgenic AD model mice were purchased from Southern Model Biotechnology Co., Ltd., which were 4 months in age, and 24-26 g in weight. The AD mice were randomly divided equally into 7 groups of 10 mice each, and the 7 groups were:

[0156] A: AD model group, as a negative control group (administration: saline);

[0157] B: cGAMP group, as a positive control group (dose: 10 mg/kg);

[0158] C: ZncGAMP group, (dose: 10 mg/kg);

[0159] D: ZncGAMP-Lipo group, (dose: 10 mg/kg);

[0160] E: complex IX (dose: 10 mg/kg);

[0161] F: complex VII (dose: 10 mg/kg); and

[0162] G: complex X (dose: 10 mg/kg).

[0163] Drugs under test:

[0164] STING agonist (cGAMP);

[0165] STING agonist metal complex (ZncGAMP);

[0166] STING agonist liposome complex (ZncGAMP-Lipo);

[0167] ZncGAMP-anti-TfR1-liposome (complex IX);

[0168] ZncGAMP-gH625-liposome (complex VII); and

[0169] ZncGAMP-gH625-PT8-liposome (complex X).

[0170] Properties: white powder.

[0171] Solvent: physiological saline.

[0172] Preparation method: prepare a solution with physiological saline solution to the required concentration before use.

[0173] Administration dose: 10 mg/kg.

[0174] Administration route: intraperitoneal injection.

[0175] Frequency: once a day for consecutive 60 days.

[0176] Morris water maze experimental device and a method thereof were described below.

[0177] A circular pool was designed, which was 1 m in diameter, 50 cm in height, 30 cm deep, and white bottom. The temperature of water in the circular pool was maintained at 23±2° C. Four equidistant points N, E, S, W marked on the wall of the circular pool as the starting point of the experiment, and divided the circular pool into four quadrants. A platform was placed in the center of the third quadrant (the platform was at an equal distance from the center of the circle of the wall of the circular pool), and submerged under the water for 1 cm to make the platform non-visible. The circular pool was surrounded by abundant reference cues (triangles, quadrangles, circles, and diamonds of different colors placed in each quadrant) and kept constant for the mice to locate the platform. Positioning navigation test was performed. The test lasted for 6 days, and the training was scheduled four times a day at a fixed period. At the beginning of the training, the platform was placed in the first quadrant and the mice were placed into the circular pool facing the wall from any one of the four starting points on the wall. A free video recording system was used to record the time for finding the platform and the swimming path of the mice. The mice were placed into the water from four different starting points (different quadrants) after the four trials. After the mice found the platform or could not find the platform within 90 seconds (the latency period was recorded as 90 seconds), they were guided to the platform by the experimenter and rested on the platform for 10 seconds before the next trial.

[0178] Space exploration tests were described below.

[0179] 24 h after the end of the positioning navigation test, the platform was removed. Then the mice were put into the water from the third quadrant, and the swimming paths of the mice were recorded within 180 s. The residence time of the mice in the target quadrant (third quadrant) and the number of times they crossed the location of the original platform were recorded to observe the spatial orientation ability of the mice. SPSS10.0 software was used for processing, and one-way ANOVA test was used to compare the significance of the differences among the groups. The experimental results were shown in Table 9 (group A: AD model control group, group B: cGAMP administration group, group C: ZncGAMP administration group, group D: ZncGAMP-Lipo administration group, group E: complex IX administration group, group F: complex VII administration group, and group G: complex X administration group). The results showed that STING agonist cGAMP, the metal complex (i.e. ZncGAMP) and the immune agonist complexes significantly improved cognitive performance of mice suffering from Alzheimer's disease after administration for 60 days. The metal complex ZncGAMP showed a superior performance to cGAMP. The three novel immune agonist complexes were more effective than the non-targeted ZncGAMP-Lipo and more significantly superior to the STING agonist cGAMP and the metal complex thereof ZncGAMP.

TABLE-US-00009 TABLE 9 Cognitive improvement effects of novel immune agonist complexes on mice suffering from Alzheimer's disease Percentage of time to cross the third quadrant Groups platform in AD mice (%) A 0.41 ± 0.11 B 0.55 ± 0.10 C 0.62 ± 0.09 D 0.75 ± 0.10 E 0.85 ± 0.08 F 0.82 ± 0.11 G 0.88 ± 0.10

Example 9 Effect of Immune Agonist Complexes on Brain Amyloid Plaques in AD Mice

[0180] Seven groups of AD mice in the above examples were tested in view of reduction of amyloid plaques in the brains of AD mice after 60 days of administration. The experiment was a thioflavin S staining experiment, and the experimental procedures were described below. 60 days after drug administration, brain tissue of mice was taken, fixed, embedded with paraffin, sectioned, de-waxed with xylene, dehydrated with ethanol gradient, and rinsed with TBS solution three times. After that, 0.3% thioflavin S (dissolved in 50% ethanol) was dropped on the tissue followed by incubation for 10 min at room temperature, rinsing with 50% ethanol three times, rinsing with TBS solution, drying in the shade, and sealing. Then the issue was examined by laser confocal microscopy (Leica, Germany) to detect the changes in the amount of amyloid plaque deposition in the brain of AD mice. The results were shown in Table 10. The results showed that STING agonist cGAMP, the metal complex ZncGAMP, and the immune agonist complexes significantly mitigated the level of amyloid plaques in the brain tissue of AD mice after 60 days of administration. The metal complex ZncGAMP was significantly more than cGAMP. The three immune agonist-brain-targeted complexes were more effective than the non-targeted ZncGAMP-Lipo and more significantly superior to the STING agonist cGAMP and the metal complex ZncGAMP.

TABLE-US-00010 TABLE 10 Effect of novel immune agonist complexes on inhibition of brain amyloid plaques in AD mice Average count points of fluorescence intensity of amyloid plaques in brain Groups tissues of AD mice (field of view area) A (AD model control group) 2900 ± 180 B (cGAMP 1800 ± 160 administration group) C (ZncGAMP 1400 ± 170 administration group) D (ZncGAMP-Lipo  800 ± 110 administration group) E (complex IX 450 ± 65 administration group) F (complex VII 500 ± 82 administration group) G (complex X 380 ± 68 administration group)

Example 10 Expression Regulation of TREM2 and Inflammatory Factor of Brain Tissues of AD Mouse by Novel Immune Agonist Complexes

[0181] ELISA kits were purchased from Cloud Clone. Flow cytometry nanobodies were purchased from eBiosciences. RT-PCR reagents were purchased from Takara. Immune fluorescence and immune histochemistry nanobodies were purchased from Abcam. Other reagents were purchased from Sigma-Aldrich. Primer synthesis was done by Shanghai Biotechnology Co. Laser confocal microscope was purchased from Leica (Germany). 7500 quantitative PCR instrument was purchased from Applied Biosystems ABI (USA). Light microscope was purchased from Leica (Germany). Enzyme marker was purchased from Hangzhou Aosheng Company. Attune flow cytometer was purchased from Thermo Fisher Scientific. RT-PCR in vitro TREM2 content was determined, and the TREM2 primer sequences were as follows:

TABLE-US-00011 Forward: (SEQ ID NO: 3) 5′-AGAAGCGGAATGGGAGC-3′; and Reverse: (SEQ ID NO: 4) 5′-GAGGTGGGTGGGAAGGA-3′.

[0182] Whether mRNA levels of TREM2 in brain tissues of mice were altered after administration of the immune agonist and complexes thereof was determined. As shown in Table 11, the B-G drug groups had significant effects on TREM2 expression levels in brain tissues of AD mouse respectively. The results showed that there was a significant increase in TREM2 expression after 60 days of administration immune agonists and complexes thereof, and the three novel immune agonist complexes were more effective than the non-targeted ZncGAMP-Lipo and more significantly superior to the STING agonist cGAMP and the metal complex ZncGAMP.

[0183] To explore the effect of the above immune agonists and complexes thereof on neuroinflammation in the brain of AD mice, the content of pro-inflammatory factors in brain tissues of AD mice was determined by ELISA method. The content of pro-inflammatory factors IL-1β and TNF-α in brain tissues of AD mice were measured respectively.

[0184] Brain tissue samples were processed through the following steps.

[0185] Fresh brain tissues of mice were taken and weighed, and the content of inflammatory factors IL-1β and TNF-α in brain tissues was determined by using the Cloud Clone ELISA kit. As shown in Table 11, the levels of pro-inflammatory factors IL-1β and TNF-α in brain tissues of mice were significantly decreased after two months of administration, compared to the levels of pro-inflammatory factors IL-1β and TNF-α in the AD model group. These results suggested that immune agonists and complexes thereof were capable of significantly reducing pro-inflammatory factors in the central nervous system. The three immune agonist-brain-targeted nanocarrier complexes (namely, complexes IX, VII, and X) were more effective than the non-targeted ZncGAMP-Lipo and more significantly superior to the STING agonist cGAMP and the metal complex thereof ZncGAMP. Therefore, the immune agonist-brain-targeted nanocarrier complexes were able to eliminate the chronic neuroinflammation present in the course of AD in mice.

[0186] Increasing evidence suggested that high levels of expression of multiple pro-inflammatory factors were closely associated with cognitive deficits in APP/PS1 di-transgenic AD mice, suggesting that a range of inflammatory signaling pathways were involved in the course of AD disease ultimately leading to cognitive deficits. Therefore, the immune agonist-brain-targeted nanocarrier complexes leaded to the reduced expression of pro-inflammatory factor, improving learning memory of mice and reducing Aβ deposition in the brain of AD mice, which was consistent with the results of the above embodiment studies.

TABLE-US-00012 TABLE 11 Modulation of TREM2 and inflammatory factors IL-1β and TNF-α in brain tissues of AD mice by immune agonist complexes Groups TREM2 (pg/mg) IL-1β (pg/mg) TNF-α (pg/mg) A (AD model 48.42 ± 0.35 58.29 ± 0.33 44.18 ± 0.09 control group) B (cGAMP 55.38 ± 0.27 46.18 ± 0.09 35.52 ± 0.35 administration group) C (ZncGAMP 62.29 ± 0.19 39.31 ± 0.17 29.41 ± 0.53 administration group) D (ZncGAMP-Lipo 71.16 ± 0.31 25.45 ± 0.41 18.32 ± 0.31 administration group) E (complex IX 80.34 ± 0.34 15.27 ± 0.23 11.19 ± 0.45 administration group) F (complex VII 78.45 ± 0.27 18.38 ± 0.31 13.32 ± 0.12 administration group) G (complex X 86.32 ± 0.19 12.24 ± 0.53  9.21 ± 0.23 administration group)

Example 11 Evaluation of Therapeutic Effect of Novel Immune Agonist Complex on Ischemic Cerebrovascular Disease

[0187] Healthy male ICR mice, weighing 18-20 g, were purchased from Shanghai Slaughter Laboratory Animal Co (quality certificate number (SCXK(SH) 2007-0005)) and raised in a cleaning experimental animal room.

Mouse model.

[0188] Local cerebral ischemia model was made by wire bolus method to verify the therapeutic effects of immune agonist complexes on cerebral ischemic brain diseases in experimental animals.

Experimental Methods

[0189] Experimental mice were anesthetized with 10% chloral hydrate intraperitoneally, and a median cervical incision was made to separate and ligate the proximal segment of the right common carotid artery, the carotid artery and branch vessels thereof. The right internal carotid artery was isolated, the pterygopalatine artery was isolated downward along the internal carotid artery, and the root was ligated for this branch. The proximal end of the internal carotid was provided with a prepared wire, and the distal end of the internal carotid was provided with an artery clip. The common carotid artery was incised at the bifurcation and tethered with the nylon line. The nylon line was tethered into the internal carotid artery, the skull and the anterior cerebral artery to block all sources of blood flowing in the cerebral arteries in the brain. The artery clip was withdrawn, and tethered with the prepared wire to suture the skin. Then the mice were returned to cage for feeding and raising. Drugs were administered by intraperitoneal injection after ischemia for 2 h, and the nylon wire was removed for reperfusion for 8 h and then drug administration. Behavioral scoring was performed after surgery, and the scoring was single-blinded with reference to the Zea Longa 5-point scale, which was scored as follows: 0, normal mice with no signs of neurological injury; 1, unable to fully extend the contralateral front paw; 2, turning in a circle to the outside; 3, relatively measured tipping; and 4, unable to walk spontaneously, and loss of consciousness injury.

Drug Administration and Effects Thereof on Behavioral Scores in a Cerebral Ischemia Model of Mice

Dose Design

[0190] The mice were randomly and equally divided into 7 groups, and 10 mice in each group, and the 7 groups were:

[0191] A: AD model group, as a negative control group (saline group);

[0192] B: cGAMP group, as a positive control group (dose: 10 mg/kg);

[0193] C: ZncGAMP group (dose: 10 mg/kg);

[0194] D: ZncGAMP-Lipo group (dose: 10 mg/kg);

[0195] E: complex IX group (dose: 10 mg/kg);

[0196] F: complex VII group (dose: 0 mg/kg);

[0197] G: complex X group (dose: 10 mg/kg).

[0198] Properties: white powder.

[0199] Solvent: physiological saline.

[0200] Preparation method: prepare the solution with physiological saline solution to the required concentration before use.

[0201] Administration dose: 10 mg/kg.

[0202] Administration method: intraperitoneal injection; 1 dose after 2 hours of ischemia, and 1 dose after 8 hours of reperfusion by pulling out the nylon line; and 1 dose after 24 hours, and 7 days of administration.

Experimental Results

[0203] The results after administration showed that intraperitoneal administration improved behavioral scores in mice with localized cerebral ischemia, and the new immune agonist complexes (IX, VII, and X) all had significantly improved efficacy over the immune agonists alone. The scoring results were shown in Table 12.

TABLE-US-00013 TABLE 12 Effect of drug administration on behavioral scores in mice with localized cerebral ischemia Behavioral scores (24 Behavioral scores (7 Groups hours after surgery) days after surgery) A (cerebral ischemia 2.90 ± 0.32 2.82 ± 0.28 model control group) B (cGAMP group) 2.26 ± 0.40 1.65 ± 0.34 C (ZncGAMP group) 2.08 ± 0.51 1.58 ± 0.36 D (ZncGAMP-Lipo 1.84 ± 0.21 1.46 ± 0.32 group) E (complex IX group) 1.75 ± 0.49 1.29 ± 0.17 F (complex VII 1.78 ± 0.42 1.36 ± 0.21 group) G (complex X group) 1.62 ± 0.34 1.25 ± 0.18

Example 12 Evaluation of Acute Toxicity of Novel Immune Agonist Complexes

Experimental Materials

[0204] 60 ICR mice (purchased from Shanghai Slaughter Laboratory Animal Co., Ltd. [Laboratory Animal Quality Certificate No. SCXK (Shanghai) 2007-0005]), were bisexual each half, 20-22 g in weight, fed with pellet diet, and fed and watered freely.

Experimental Methods

[0205] ICR mice were injected intraperitoneally with 1 g/kg of the novel immune agonist complexes (I, II, III, IV, V, VI, VII, VIII, IX, X) (prepared with PBS), and the toxic reactions and mortality of the mice were observed within 14 days after the administration. The results showed that the mice moved normally after intraperitoneal injection of the drug, and none of the mice died within 14 days after the administration. On the 15.sup.th day, all mice were executed, dissected to be examined visually, and no obvious lesions were found.

Experimental Results

[0206] The above experimental results showed that the maximum tolerated dose (MTD) was not less than 1 g/Kg when administered intraperitoneally, indicating that the acute toxicities of the novel immune agonist complexes were low.