CN112957475A - Composition for preventing and/or treating tumors and application thereof - Google Patents

The invention belongs to the technical field of biological medicines, and particularly relates to a composition for preventing and/or treating tumors and application thereof. A composition for the prevention and/or treatment of tumors therein, comprising one or more TLR agonists, one or more TNFRSF agonists and one or more STING agonists. The invention innovatively combines the TLR agonist, the TNFRSF agonist and the STING agonist, and simultaneously, the TNFRSF agonist, the STING agonist and the STING agonist are applied in local tumors, so that the TNFRSF agonist can induce strong anti-tumor immune response to cause the regression of tumors at injection sites and non-injection sites.

Composition for preventing and/or treating tumors and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a composition for preventing and/or treating tumors and application thereof.

Background

According to the data published by the world health organization, cancer poses the greatest burden worldwide. Malignant tumors can be considered as the primary public health care problem, which puts a huge burden on the clinic, disturbs social standards, and erodes a large amount of economic resources. Although the incidence and mortality of malignant tumors has increased year by year, most malignant tumors lack an effective treatment regimen, and the traditional treatment modalities are still limited to radiation therapy, chemotherapy, and surgical resection. In fact, less than 30% of patients with tumors receive the opportunity for surgical treatment, while many patients experience severe adverse effects immediately or chronically after receiving systemic or local chemotherapy, and in some cases, adverse effects associated with treatment outweigh the benefits of treatment, or even worsen the patient's condition. Radiotherapy, chemotherapy and surgery have significant limitations in the treatment of tumors and are difficult to cure malignant tumors, especially metastatic malignant tumors.

In recent years, immunotherapy has become a hot research topic for tumor therapy. Monoclonal antibodies against immune checkpoint cytotoxic T lymphocyte-associated protein 4(CTLA-4) and programmed cell death protein 1(PD-1) have shown significant therapeutic efficacy and have been approved by the FDA for cancer treatment. In addition, chimeric antigen receptor-T (CAR-T) cell therapy has been approved for the treatment of certain hematological malignancies. Tumor immunotherapy has made more and more breakthrough progress. At present, the main tumor immunotherapy means are agonism or blockade therapy, adoptive cell therapy, genetic engineering cell therapy, tumor vaccine, etc. directed to immune checkpoints. These approaches, while each are well developed, tumor immunotherapy still faces a number of obstacles due to the complexity of the tumor microenvironment and the refractory nature of metastatic tumors.

Currently, tumor immunotherapy still faces three major challenges:

problem 1. in most solid tumors, the tumor immunogenicity is low. Dendritic Cells (DCs) are the most powerful Antigen Presenting Cells (APCs) that induce the formation of specific Cytotoxic T Lymphocytes (CTLs). However, in the tumor microenvironment, DCs are mostly immature phenotypes, poorly active, unable to present tumor antigens to effector T cells, and unable to initiate anti-tumor immune responses. This "priming disability" leads to limited tumor immune efficacy.

Problem 2. single immunotherapy or immunotherapy drugs do not achieve the expected effect. Genome-wide analysis has shown that heterogeneity between tumor-bearing individuals is very significant, which results in large differences in the responsiveness of different patients to the same monotherapy, and unsatisfactory results are not achieved. For example, while immune checkpoint inhibitors can block the inhibitory effects of cell contact-dependent protein pathways on effector T cells for pancreatic cancer, there are many soluble immunosuppressive factors that inhibit the function of effector T cells in the tumor microenvironment of pancreatic cancer. Pancreatic cancer, a typical "cold" tumor, contains a large number of dense stromal cells and few Tumor Infiltrating Lymphocytes (TILs), leading to the failure of some immune checkpoint inhibitors. This forces tumor immunotherapy to shift from monotherapy directed at a single target or single pathway to a combination of multi-target and multi-pathway approaches.

Problem 3. immune toxic reaction caused by systemic immune activation. Due to the systemic administration of immunotherapy, the systemic immune system of the subject is rapidly activated, which on the one hand causes an objective response against the tumor and on the other hand also brings about an autoimmune adverse reaction that is difficult to recognize. For example, the gastrointestinal system is one of the organ systems most commonly affected by autoimmune inflammation. The incidence of autoimmune enterocolitis in patients receiving tumor immunotherapy varies from 1% to 25%, depending on the type of drug the patient receives. Systemic administration inevitably over-activates systemic immunity, resulting in normal tissue involvement. This forces the tumor immunotherapy to switch to local, precise dosing.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art and provide a composition for preventing and/or treating tumors and application thereof.

The technical scheme adopted by the invention is as follows: a composition for the prevention and/or treatment of a tumor comprising one or more TLR agonists, one or more TNFRSF agonists and one or more STING agonists.

The TLR agonist is a TLR9 agonist.

The TLR9 agonist is an oligonucleotide and/or hemozoin; the oligonucleotides are cytosine-phosphorothioate-guanine oligodeoxynucleotides containing unmethylated cytosine and guanine in class A, class B or class C.

The TNFRSF agonist is an OX-40 agonist.

The OX-40 agonist is one or more of an OX-40 agonist antibody, an OX40L agonist fragment, an OX-40 oligomeric receptor, and OX-40 immunoadhesin.

The OX-40 agonist is a trimeric OX40L-Fc protein, which trimeric OX40L-Fc protein comprises a fragment of one or more extracellular domains of OX 40L.

The STING agonist is a cyclic dinucleotide or a derivative thereof.

The cyclic dinucleotide is one or more of cyclic adenosine di-phosphate monophosphate, cyclic guanosine di-phosphate monophosphate, cyclic inosine monophosphate, cyclic guanosine monophosphate adenosine monophosphate, or cyclic adenosine monophosphate-inosine monophosphate and cyclic guanosine monophosphate-inosine monophosphate.

The use of a composition for the prevention and/or treatment of a tumour as described above in the manufacture of a medicament for the treatment or prevention of a tumour.

A medicament or a kit, characterized in that the medicament or the kit contains the composition for preventing and/or treating tumor.

The invention has the following beneficial effects: the invention innovatively combines the TLR agonist, the TNFRSF agonist and the STING agonist, and simultaneously, the TNFRSF agonist, the STING agonist and the STING agonist are applied in local tumors, so that the TNFRSF agonist can induce strong anti-tumor immune response to cause the regression of tumors at injection sites and non-injection sites.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for those skilled in the art to obtain other drawings based on the drawings without inventive exercise.

In FIG. 1, (A) is a graph of the tumor volume at the injection site after different treatments for groups A-E of cervical cancer-bearing mice, demonstrating the antitumor ability of CPG + OX40+ cGAMP; (B) for the plot of the volume of the uninjected tumors after different treatments of cervical cancer-bearing mice in the A-E group, the results showed that only CPG + OX40+ cGAMP treatment was able to produce distant tumoricidal ability, confirming the superiority of CPG + OX40+ cGAMP therapy for metastatic tumors.

FIG. 2 is a graph of mouse survival following differential treatment of groups A-E cervical cancer-bearing mice, showing that CPG + OX40+ cGAMP significantly extends survival in tumor-bearing mouse models.

FIG. 3 is a graph showing the body weight change of mice treated differently with group A-E cervical cancer-bearing mice, demonstrating that the body weight of mice treated with CPG + OX40+ cGAMP is not different from that of mice treated with blank treatment, i.e., the therapy of the present invention has no obvious toxic side effects when applied to tumors.

In FIG. 4, CD3, CD4 and CD8 cell infiltrates of the non-injected tumors of mice treated differently with mice bearing cervical cancer of groups A-E. It was confirmed that CPG + OX40+ cGAMP treatment caused a large increase in the number of tumor-infiltrating lymphocytes at the site of non-injection, with the most significant increase in CD 4-positive lymphocytes, and thus the triple group had the best antitumor effect.

In FIG. 5, (A) is a plot of the tumor volume at injection after different treatments in groups A-E melanoma-bearing mice, again demonstrating the antitumor ability of CPG + OX40+ cGAMP. (B) Is a tumor volume map of the non-injected part of a group A-E melanoma tumor-bearing mice after different treatments, and the result is similar to the experimental result of the cervical cancer tumor-bearing mice: only CPG + OX40+ cGAMP treatment was able to produce distant tumoricidal capacity, confirming the superiority of CPG + OX40+ cGAMP therapy for metastatic tumors.

FIG. 6 is a graph showing the survival of mice treated differently with melanoma-bearing mice of groups A-E, similar to the experimental results for mice bearing cervical cancer: only CPG + OX40+ cGAMP treatment significantly extended survival in tumor-bearing mouse models.

In FIG. 7, (A) shows the tumor volume of melanoma tumor-bearing mice treated by injection with combinations of CpG + cGAMP and different immunodetection point inhibitors, demonstrating that CpG + cGAMP in combination with different immunodetection point inhibitors all have anti-tumor effects in situ. (B) Tumor volumes were examined in untreated sites after injection treatment of melanoma-bearing mice with combinations of CpG + cGAMP and different immunodetection point inhibitors, demonstrating that only CpG + OX40+ cGAMP treatment was able to produce a distant tumor killing capability, demonstrating the superiority of CpG + OX40+ cGAMP therapy for metastatic tumors.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

A composition for the prevention and/or treatment of a tumor comprising one or more TLR agonists, one or more TNFRSF agonists and one or more STING agonists.

The invention employs a TLR agonist which, after being administered locally within a tumor, is specifically recognized by dendritic cells, activates and mediates maturation of dendritic cells. The costimulation molecules on the surface of the dendritic cells are up-regulated, the antigen presenting capability is enhanced, and simultaneously, the dendritic cells generate more interferon-alpha to promote the maturation of the marrow dendritic cells, so as to indirectly activate T cells. The STING agonist is used, and after the agonist is applied, the STING agonist leads to the activation of STING pathway of dendritic cells in a tumor microenvironment, promotes antigen cross presentation, increases the expression of CCR7 on the dendritic cells, and improves the transportation of homing effector T cells of antigen presenting cells by various Th1 cytokines. Therefore, the dendritic cells are used as antigen presenting cells with the strongest functions to be activated and proliferated in a tumor microenvironment, and the antigen presenting capacity is greatly enhanced, so that the problem of low tumor immunogenicity is solved.

The invention combines three immune targets or channels simultaneously, and changes from a single therapy to a mode of combining multiple targets and multiple channels. First, Toll-like receptors (TLRs) are present on many cells of the immune system and have been shown to be involved in the innate immune response, which is a key means for vertebrates to recognize and establish immune responses to foreign molecules, to recognize pathogen-associated molecular patterns of bacteria, fungi, parasites and viruses, and to provide a means to link innate and adaptive immune responses. The invention uses TLR agonist which can activate NF-kB signal and stimulate IFN-a secretion; the TNFRSF agonist is used, so that the survival time of T cells can be prolonged, the killing capacity of the T cells can be promoted, and the immunosuppressive action in a tumor microenvironment can be improved; the STING agonist used in the invention stimulates the induction of INF-beta through a STING pathway, mediates the activation of NF-kappa B and IRF-3, and then starts the transcription of INF-beta gene to generate beta-interferon. In conclusion, the combination of the agonists of the three types of targets or pathways can effectively solve the problem of tumor heterogeneity.

The invention can be applied in local tumor, avoids systemic medication, achieves the purpose of treating tumor at the injection site by modifying local tumor microenvironment, simultaneously causes tumor decline at the non-injection site by improving tumor immunogenicity, modifying tumor infiltration cell types, cytokines and other factors, and avoids the occurrence of systemic autoimmune toxic and side effects.

In some embodiments of the invention, the TLR agonist is a TLR9 agonist, and a common TLR9 agonist includes an oligonucleotide that is a cytosine-phosphorothioate-guanine oligodeoxynucleotide containing unmethylated cytosine and guanine, including class a (also referred to as class D), class B (also referred to as class K), and class C, and hemozoin. Class A consists of a PO backbone centered on a sequence containing a CpG palindrome and a PS-modified 3' -poly-G strand. CpG-A ODNs stimulate plasmacytoid dendritic cells (pDCs) to produce IFN-a more strongly, but activate TLR 9-dependent NF-kB signaling pathway less strongly, producing less pro-inflammatory cytokines (e.g., interleukin 6). Class B is the complete PS backbone structure sequence containing one or more CpG dinucleotides. CpG-B ODNs can strongly activate B cells and TLR 9-mediated NF-kB signals, but have weak effect on stimulating IFN-a secretion. The Class C structure is a mixture of classes A and B. It contains the complete PS backbone and CpG palindrome sequences. CpG-C ODNs can strongly cause pDC to secrete IFN-a and can also obtain better effect when B cells are stimulated. In some embodiments of the invention, the C-class TLR9 agonist is specifically selected as CpG ODN 2395, and the sequence of the CpG ODN 2395 is 5'-TCGTCGTTTTCGGCGCGCGCCG-3'.

In some embodiments of the invention, the TNFRSF agonist is selected from OX-40 agonists, and the OX-40 agonists are selected from OX-40 agonist antibodies, OX40L agonist fragments, OX-40 oligoreceptors, and OX-40 immunoadhesins. In some embodiments of the invention, an OX-40 agonist antibody is specifically selected, wherein the OX-40 agonist antibody belongs to a member of tumor necrosis factor superfamily, is an agonist receptor expressed on the cell surface of activated CD4 positive T cells and CD8 positive T cells, the signal of the OX-40 agonist antibody can activate downstream NF-kappa B, PI3K and PKB pathways, the continuous activation of the pathways can finally prolong the survival time of the T cells, expand the memory of the T cells and promote the killing capacity of the T cells, and in addition, the OX-40 agonist antibody can improve the immunosuppressive effect in a tumor microenvironment by inhibiting the differentiation and activity of regulatory T cells (Tregs) and further enhance the functions of effector T cells. In some embodiments of the invention, the OX-40 agonist antibody is specifically selected from a trimeric OX40L-Fc protein comprising a fragment of one or more extracellular domains of OX40L, or a full-length human IgG1 antibody.

In some embodiments of the invention, the STING agonist is specifically selected from a cyclic dinucleotide or a derivative thereof, wherein the cyclic dinucleotide is cyclic di-adenosine monophosphate (CDA), cyclic di-guanosine monophosphate (CDG), cyclic di-inosine monophosphate (CDI), cyclic guanosine monophosphate (cGAMP), cyclic adenosine monophosphate-inosine monophosphate or cyclic guanosine monophosphate inosine monophosphate, and in some embodiments of the invention, cyclic guanosine monophosphate (cGAMP) is selected. cGAMP, a STING agonist, is a cytoplasmic DNA sensor that acts as a second messenger to stimulate the induction of INF-beta through the STING pathway, mediate the activation of NF-. kappa.B and IRF-3, and then initiate transcription of the INF-beta gene, producing beta interferon.

The application of the composition for preventing and/or treating tumors in the preparation of medicines for treating or preventing tumors. The composition for preventing and/or treating tumors provided by the invention can be used as an active ingredient to be combined with a pharmaceutically acceptable carrier to prepare a composite biological preparation as a medicinal preparation for preventing and/or treating tumors.

A kit for preventing and/or treating tumor comprising the composition for preventing and/or treating tumor as described above, wherein one or more TLR agonist, one or more TNFRSF agonist and one or more STING agonist are mixed as a single agent or separately provided as a plurality of agents. Agonists of the three targets or pathways may be combined into a complex biological formulation for administration to a localized tumor, or may be administered separately and simultaneously to provide enhanced disease treatment or prevention. Other chemical and/or biological therapeutic agents may also be included in the kit, such as anti-angiogenic agents, growth inhibitory agents, other anti-neoplastic agents currently known or combinations thereof.

The following is a sample preparation process used in some embodiments of the invention.

Culture of TC-1 cervical cancer cell line

TC-1 murine cervical carcinoma cells were cultured in RPMI 1640 complete medium containing 10% heat-inactivated fetal bovine serum, penicillin 100U/mL, streptomycin 100U/mL, cultured in a 5% CO2 incubator at 37 ℃ with the culture medium changed daily and digested with 0.1% trypsin every 3 days for passaging. When the cells are in the logarithmic growth phase, digesting the cells, centrifuging at 1000 r/min, counting the number of living cells, and then suspending in balanced salt solution PBS to prepare suspension containing 4 x 10^6 cervical cancer cells per 1 mL.

Secondly, establishing a TC-1 cervical carcinoma C57BL/6 tumor-bearing mouse model

30 healthy inbred C57BL/6 mice, female, SPF grade, 6-8 weeks, body weight (20. + -.2) g. The temperature of the breeding environment is 22-25 ℃, the humidity is 45-50%, the illumination period is 12h illumination/12 h darkness, and water is freely drunk/eaten. The replacement of the feed, the drinking water and the padding is carried out once every 3 days by the mice, wherein the feed, the drinking water and the padding required for raising the mice are all subjected to autoclaving treatment. Mice were acclimatized for one week prior to experimental treatment. And (2) inoculating the cultured TC-1 tumor cells to the left groin of a normal mouse subcutaneously (each mouse is inoculated with 2 x 10^5 cells), performing the same operation on the right groin of the mouse after two days to construct a bilateral cervical cancer tumor animal model, and successfully modeling when the left groin tumor grows to 20-30mm 2.

Culture of murine melanoma cell line B16

B16 murine melanoma cell line was cultured in DMEM complete medium containing 10% heat-inactivated fetal bovine serum, penicillin 100U/mL, streptomycin 100U/mL, cultured in a 5% CO2 incubator at 37 ℃ with the medium changed daily and digested with 0.1% trypsin every 3 days. When the cells are in the logarithmic growth phase, the cells are digested, centrifuged at 1000 r/min, the number of the living cells is counted and then the living cells are resuspended in a balanced salt solution PBS to prepare a suspension containing 4 multiplied by 10^6 melanoma cells per 1 mL.

Fourthly, establishing a B16 melanoma C57BL/6 tumor-bearing mouse model

30 healthy inbred C57BL/6 mice, female, SPF grade, 6-8 weeks, body weight (20. + -.2) g. The temperature of the breeding environment is 22-25 ℃, the humidity is 45-50%, the illumination period is 12h illumination/12 h darkness, and water is freely drunk/eaten. The replacement of the feed, the drinking water and the padding is carried out once every 3 days by the mice, wherein the feed, the drinking water and the padding required for raising the mice are all subjected to autoclaving treatment. Mice were acclimatized for one week prior to experimental treatment. The cultured B16 melanoma cells are inoculated to the left groin of a normal mouse through subcutaneous inoculation (each mouse is inoculated with 2 x 10^5 cells), the same operation is carried out on the right groin of the mouse after two days, a melanoma bilateral tumorigenesis animal model is constructed, and when left groin tumor grows to 20-30mm2, the molding is successful.

The following are some specific examples of the present invention.

Example 1 grouping and administration of TC-1 cervical carcinoma C57BL/6 tumor-bearing mouse models

The following are observations from the different test groups described above:

The A-E group of tumor-bearing mice were administered intratumorally to the left inguinal: tumor volume sizes were observed and recorded for the a-E group at injection (left groin) and non-injection (right groin) following PBS treatment, CPG + OX40 treatment, cGAMP treatment, CPG + OX40+ cGAMP treatment. The results are shown in FIG. 1 (A): for the injected tumors, the CPG group and CPG + OX40+ cGAMP treated group were the best and there was no significant statistical difference between the two. The tumor volumes of the groups A-E tumor-bearing mice at the non-injection sites are shown in FIG. 1 (B), and the results show that: only after CPG + OX40+ cGAMP treatment, growth inhibition of the non-injected tumors appeared, and none of the remaining groups appeared. This confirms the excellent efficacy of the present invention against metastatic tumors.

The A-E group of tumor-bearing mice were administered intratumorally to the left inguinal: survival of group a-E tumor-bearing mice was observed and recorded following PBS treatment, CPG + OX40 treatment, cGAMP treatment, CPG + OX40+ cGAMP treatment. The results are shown in FIG. 2: the CPG + OX40+ cGAMP treatment group remarkably prolongs the survival period of the tumor-bearing mouse model, the CPG + cGAMP treatment has the effect of slightly prolonging the survival period, and the rest groups have no obvious benefit on the survival period of the tumor-bearing mice.

The A-E group of tumor-bearing mice were administered intratumorally to the left inguinal: after PBS treatment, CPG + OX40 treatment, cGAMP treatment, CPG + OX40+ cGAMP treatment, group a-E tumor-bearing mouse body weights were observed and recorded. The results are shown in FIG. 3: abnormal performance does not appear in all groups of mice, and the body weight of the mice in the B-E group and the body weight of the mice in the blank control A group do not have statistical difference, namely: after the mice are administrated with CPG + OX40+ cGAMP in tumors, no obvious toxic or side effect occurs, the general condition is good, and no abnormal expression occurs.

The A-E group of tumor-bearing mice were administered intratumorally to the left inguinal: after PBS treatment, CPG + OX40 treatment, cGAMP treatment, CPG + OX40+ cGAMP treatment, the tumor-infiltrated CD3, CD4 and CD8 cells of the tumor-bearing mice in the A-E group, which is not injected, were detected by flow cytometry. The results are shown in FIG. 4: the tumors at the injection sites were treated with CPG, CPG + OX40 and CPG + OX40+ cGAMP, which all caused an increase in the number of tumor-infiltrating lymphocytes at the non-injection sites, but the increased number of CD 4-positive T lymphocytes in each group was most significant in the CPG + OX40+ cGAMP-treated group, and thus the treatment effect was the best.

Example 2 melanoma B16 mice model C57BL/6 bearing tumor were grouped and treated with different doses

The following are observations from the different test groups described above:

The A-E group of tumor-bearing mice were administered intratumorally to the left inguinal: tumor volume sizes were observed and recorded for the a-E group at injection (left groin) and non-injection (right groin) following PBS treatment, CPG + OX40 treatment, cGAMP treatment, CPG + OX40+ cGAMP treatment. Results as shown in fig. 5, the results of the melanoma animal experiments were similar to cervical cancer: for the injected tumors, the CPG group and CPG + OX40+ cGAMP treated group were the best and there was no significant statistical difference between the two. Similarly, the tumor volumes of the groups A-E tumor-bearing mice at the non-injection sites are shown in FIG. 6, and the results show that: only after CPG + OX40+ cGAMP treatment, growth inhibition of the non-injected tumors appeared, and none of the remaining groups appeared. This demonstrates the excellent efficacy of the present invention on metastatic tumors without differentiating between cancer species.

In conclusion, the anti-tumor composition or method containing the TLR agonist, the TNFRSF agonist and the STING agonist has a strong immunotherapy effect on tumors, modifies a tumor microenvironment to generate an anti-tumor distant vaccine effect, has an excellent curative effect on metastatic tumors, and more importantly, the anti-tumor effect obtains similar effect evidence in different cancer species, so that the anti-tumor composition or method can be applied to anti-tumor treatment without distinguishing the cancer species. Meanwhile, the invention avoids the toxic and side effects of immunotherapy caused by systemic administration and overcomes the general problem of the current tumor immunotherapy.

Example 3 grouping and administration of B16 melanoma C57BL/6 tumor-bearing mouse models

To confirm whether other drug combinations also have such systemic anti-tumor effects, we also performed various drug combination experiments. The mice were randomly divided into 9 groups of 6 mice each, and a total of 54 mice were included. Group A: PBS treatment group, 50ul PBS was injected intratumorally in the left groin of each mouse; group B: CPG + cGAMP treated groups, each mouse was intratumorally injected with CpG 2395(1-1000ug (preferably 1-200ug, as 50ug in this experiment)), cGAMP (1-1000ug (preferably 1-200ug, as 10ug in this experiment)); group C: CPG + OX40+ cGAMP treated groups, each mouse was intratumorally injected with CpG 2395(1-1000ug (preferably 1-200ug, as 50ug in this experiment)), OX40(1-1000ug (preferably 1-200ug, as 30ug in this experiment), cGAMP (1-1000ug (preferably 1-200ug, as 10ug in this experiment)); group D: CPG + LAG-3+ cGAMP treated groups, each mouse was injected intratumorally with CpG 2395(1-1000ug (preferably 1-200ug, e.g. 50ug in this experiment)), LAG-3(1-1000ug (preferably 5-200ug, e.g. 30ug in this experiment), cGAMP (1-1000ug (preferably 1-200ug, e.g. 10ug in this experiment)); group E: CPG + TIM-3+ cGAMP treated groups, each mouse was intratumorally injected with CpG 2395(1-1000ug (preferably 1-200ug, as 50ug in this experiment)), TIM-3(1-1000ug (preferably 1-200ug, as 30ug in this experiment), cGAMP (1-1000ug (preferably 1-200ug, as 10ug in this experiment)); and F group: CPG + BTLA + cGAMP treated groups, each mouse was intratumorally injected with CpG 2395(1-1000ug (preferably 1-200ug, e.g., 50ug in this experiment)), BTLA (1-1000ug (preferably 1-200ug, e.g., 30ug in this experiment)), cGAMP (1-1000ug (preferably 1-200ug, e.g., 10ug in this experiment)); group G: CPG + NKG2A + cGAMP treated groups, each mouse was intratumorally injected with CpG 2395(1-1000ug (preferably 1-200ug, e.g. 50ug in this experiment)), NKG2A (1-1000ug (preferably 1-200ug, e.g. 30ug in this experiment)), cGAMP (1-1000ug (preferably 1-200ug, e.g. 10ug in this experiment)); group H: CPG + CD28+ cGAMP treated groups, each mouse was injected intratumorally with CpG 2395(1-1000ug (preferably 1-200ug, as 50ug in this experiment)), CD28(1-1000ug (preferably 1-200ug, as 30ug in this experiment), cGAMP (1-1000ug (preferably 1-200ug, as 10ug in this experiment)); group I: the CPG +41BB + cGAMP treated group, each mouse was injected intratumorally with CpG 2395(1-1000ug (preferably 1-200ug, e.g., 50ug in this experiment)), 41BB (1-1000ug (preferably 1-200ug, e.g., 30ug in this experiment)), cGAMP (1-1000ug (preferably 1-200ug, e.g., 10ug in this experiment)). After the administration, measurement of tumor size and observation of mouse status were performed every 2 days, and tumor volume = (long diameter × short diameter) = short diameter)/2, and the observation was continued.

The following are observations from the different test groups described above:

firstly, observing the tumor volume difference between the injection site and the non-injection site of each group of melanoma

The A-I group tumor-bearing mice were administered intratumorally with the following drugs: after PBS treatment, CPG + cGAMP treatment, CPG + OX40+ cGAMP treatment, CPG + LAG-3+ cGAMP treatment, CPG + TIM-3+ cGAMP treatment, CPG + BTLA + cGAMP treatment, CPG + NKG2A + cGAMP treatment, CPG + CD28+ cGAMP treatment, and CPG +41BB + cGAMP treatment, the tumor volume sizes at the group A-I injection site (left groin) and the non-injection site (right groin) were observed and recorded. The results are shown in FIG. 7 (a): for the injected tumors, all treatment groups were significantly effective relative to the PBS group. The tumor volumes of the groups A-I tumor-bearing mice at the non-injection sites are shown in FIG. 7 (b), and the results show that: only after CPG + OX40+ cGAMP treatment, growth inhibition of the non-injected tumors appeared, and none of the remaining groups appeared. This confirms the excellent efficacy of the present invention against metastatic tumors.

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.