Combination therapy composition and combination therapy method for tumor treatment

Abstract

The invention relates to a combined treatment composition and a combined treatment method for tumor treatment. The combined treatment composition or a medicine box comprises a therapeutically effective amount of platinum chemotherapeutic drugs, a therapeutically effective amount of CpG oligonucleotide and a therapeutically effective amount of R848. The invention also provides a combined treatment method for treating tumors by using the combined treatment composition or the medicine box, which comprises the following steps of: firstly, killing the tumors by using a low-dose chemotherapy medicine to release neoantigens of the tumors, and enabling an organism immune system to autonomously recognize and screen the neoantigens; cpG oligonucleotide and an R848 immune activator are used for inducing and promoting an organism to generate tumor neoantigen specific immune response, and the anti-tumor function of T cells is improved and strengthened, so that the effect of inhibiting tumor growth is achieved.

Description

Combined therapeutic compositions and combined therapeutic methods for treating tumors Technical Field

[0001] The present invention relates to the field of medical technology, and specifically to a combined therapeutic composition and a combined therapeutic method for treating tumors. Background Art

[0002] Cancer remains one of the major diseases threatening human health in today's society. According to estimated data from the World Health Organization's International Agency for Research on Cancer (IARC), there were 19.29 million new cases of cancer worldwide in 2020, and the number of cancer deaths reached 9.96 million.

[0003] Targeted therapy combined with chemotherapy and / or radiotherapy has become an important treatment for cancer. However, chemotherapy drugs have significant side effects, and patients often forgo treatment due to intolerance. While targeted therapy has shown promising results for many tumors, it is not suitable for all tumors due to ineffectiveness caused by mutations in key tumor genes. Tumor immunotherapy has been a key research area in recent years, but immune tolerance (or immune neglect) is one of the fundamental reasons why tumor cells survive and grow uncontrollably in human tissues. Strengthening the immunogenicity of anti-tumor cells so that the immune system no longer ignores them remains a challenge and a key issue in the fight against cancer.

[0004] The increase in neoantigens generated by tumor mutations is closely associated with the formation of tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). These antigens possess enhanced immunogenicity and can increase T cell infiltration, resulting in anti-tumor effects. However, immunotherapy regimens based on neoantigens, TAAs, and TSAs suffer from a significant drawback: inconsistent therapeutic efficacy. This is due to the fact that the selected and designed antigens cannot guarantee an effective anti-tumor immune response and efficacy, and cannot keep pace with the rapid mutation of tumor antigens. The successful application of PD-1 antibodies in cancer treatment demonstrates that stimulating the body's intrinsic immune system can have anti-tumor effects, and that significant anti-tumor efficacy can be achieved simply by enhancing and awakening the function of these T cells. In situ vaccination is a promising cancer immunotherapy approach that blocks the mechanisms cancer cells use to evade immune detection, independent of specific tumor-associated antigens. This approach has the potential to elicit a broad immune response against cancer cells, leading to long-term remission or cure. However, a major limitation is that the amount of tumor antigens induced in situ is insufficient to induce a robust anti-tumor immune response and response, resulting in weak and inconsistent anti-tumor efficacy in this field. This technology needs to be greatly improved in terms of antigen release ability and activation of anti-tumor immune response.

[0005] The use of chemotherapy drugs can cause cellular DNA damage, killing some tumor cells, which helps increase the mutation load and induce the production of subclonal neoantigens. Neoantigens only provide immunogenicity for anti-tumor immunity and cannot effectively induce anti-tumor immunity in the body. Appropriate molecules are needed to activate the body's immune system.

[0006] Summary of the Invention

[0007] Based on this, the present invention further promotes the effectiveness of tumor treatment by using chemotherapy drugs to induce the production of neoantigens. This allows the tumor patient's immune system to autonomously identify and screen these neoantigens, while simultaneously enhancing and strengthening the anti-tumor function of T cells, ultimately achieving a therapeutic solution with excellent anti-tumor effects. This new therapy, which fully utilizes the tumor antigen release effect of chemotherapy during the killing process, combined with a Toll-like receptor (TLR) activator, offers a new approach to tumor treatment.

[0008] TLR activators are used in tumor treatment, currently mostly as adjuvants in personalized tumor vaccines. Some are used in combination with conventional chemotherapy drugs or immune checkpoint-targeted drugs, significantly improving tumor treatment efficacy. However, there is currently no research on drugs that combine "low-dose chemotherapy drugs with TLR activators" to enhance tumor-specific cellular immune responses. Unlike other existing immunotherapy strategies, the unique therapeutic concept of this invention is that it is not limited by tumor type or tumor antigen mutations. Through low-dose chemotherapy, tumor antigens are released, and immune activators induce specific tumor immunity in individual tumors, thereby improving tumor treatment efficacy.

[0009] The present invention innovatively utilizes the mechanism of immunological activation of TLR activators, allowing the immune system of tumor patients to autonomously identify and screen neoantigens, while simultaneously enhancing and strengthening the anti-tumor function of T cells and inhibiting the interference of the tumor immune microenvironment, thereby achieving a treatment plan with a final good anti-tumor effect. In order to further promote the therapeutic effect of tumors, the tumor antigen release effect caused by chemotherapy drugs during the killing process is fully utilized, and combined with TLR activators to activate the body's intrinsic anti-tumor immunity, the therapeutic effect of tumors is improved. The combination therapy of the present invention has innovative theory and good clinical application prospects.

[0010] In view of this, the present invention is proposed.

[0011] The purpose of the present invention is to provide a pharmaceutical composition for combined tumor treatment, and a combined treatment method for treating tumors using the combined therapeutic pharmaceutical composition, in the hope of achieving a broad-spectrum anti-tumor effect.

[0012] In order to solve the above technical problems and achieve the above objectives, the present invention provides the following technical solutions:

[0013] In one aspect, the present invention provides a pharmaceutical composition for combined treatment of tumors, comprising a therapeutically effective amount of a platinum-based chemotherapy drug, a therapeutically effective amount of a CpG oligonucleotide, and a therapeutically effective amount of R848.

[0014] In an optional embodiment, the platinum compound is selected from one or more of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin. Preferably, the platinum compound is cisplatin.

[0015] In an alternative embodiment, the therapeutically effective amount of CpG oligonucleotide (CpG ODN) is a B-class CpG ODN or a C-class CpG ODN.

[0016] Preferably, the class B CpG ODN is selected from one or more of ODN 1826, ODN 1018, and ODN 2006 / 7909.

[0017] Preferably, the C-class CpG ODN is selected from one or more of ODN M362, ODN 2395, and D-SL03.

[0018] In an optional embodiment, the therapeutically effective amount of R848 is selected from one or more of water-soluble R848 and fat-soluble R848.

[0019] In an optional embodiment, the therapeutically effective amount of the platinum chemotherapy drug is administered in a dosage of 2 μg / kg to 4000 μg / kg.

[0020] In an optional embodiment, the therapeutically effective amount of the CpG oligonucleotide is administered in a dosage of 0.001 mg / time to 32 mg / time.

[0021] In an optional embodiment, the therapeutically effective amount of R848 is administered in a dosage of 0.001 mg / time to 5 mg / time.

[0022] In an optional embodiment, the therapeutically effective amount of the platinum chemotherapy drug is administered in a dosage of 2 μg / kg to 20 μg / kg;

[0023] The therapeutically effective amount of the CpG oligonucleotide is administered in a dosage of 0.001 mg / time to 32 mg / time;

[0024] The therapeutically effective amount of R848 is administered in a dosage of 0.001 mg / time to 1 mg / time.

[0025] In a preferred embodiment, the therapeutically effective amount of the platinum chemotherapy drug is administered in a dosage of 2 μg / kg to 400 μg / kg;

[0026] The therapeutically effective amount of the CpG oligonucleotide is administered in a dosage of 0.05 mg / time to 2 mg / time;

[0027] The therapeutically effective amount of R848 is administered in a dosage of 0.01 mg / time to 1 mg / time.

[0028] In another aspect, the present invention also provides a method for treating a subject suffering from a tumor or cancer using the above-mentioned pharmaceutical composition, the method comprising administering a therapeutically effective amount of the pharmaceutical composition to the subject; the pharmaceutical composition comprises a therapeutically effective amount of a platinum chemotherapy drug, a therapeutically effective amount of a CpG oligonucleotide and a therapeutically effective amount of R848.

[0029] In an optional embodiment, the platinum compound is selected from one or more of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin. Preferably, the platinum compound is cisplatin.

[0030] In an alternative embodiment, the therapeutically effective amount of CpG oligonucleotide (CpG ODN) is a B-class CpG ODN or a C-class CpG ODN.

[0031] Preferably, the class B CpG ODN is selected from one or more of ODN 1826, ODN 1018, and ODN 2006 / 7909.

[0032] Preferably, the C-class CpG ODN is selected from one or more of ODN M362, ODN 2395, and D-SL03.

[0033] In an optional embodiment, the therapeutically effective amount of R848 is selected from one or more of water-soluble R848 and fat-soluble R848.

[0034] In an optional embodiment, the therapeutically effective amount of the platinum chemotherapy drug is administered in a dosage of 2 μg / kg to 4000 μg / kg.

[0035] In an optional embodiment, the therapeutically effective amount of the CpG oligonucleotide is administered in a dosage of 0.001 mg / time to 32 mg / time.

[0036] In an optional embodiment, the therapeutically effective amount of R848 is administered in a dosage of 0.001 mg / time to 5 mg / time.

[0037] In an optional embodiment, the therapeutically effective amount of the platinum chemotherapy drug, the therapeutically effective amount of the CpG oligonucleotide, and the therapeutically effective amount of R848 are administered to a subject in need thereof in sequence.

[0038] In an alternative embodiment, the administration time of the therapeutically effective amount of the platinum chemotherapy drug occurs before the administration time of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848.

[0039] In an alternative embodiment, the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are administered simultaneously.

[0040] In an optional embodiment, the therapeutically effective amount of the platinum chemotherapy drug is administered once, and the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are administered 1 to 4 times as one administration cycle.

[0041] In an optional embodiment, the first administration of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 is separated from the first administration of the therapeutically effective amount of the platinum chemotherapy drug by 1 to 3 days.

[0042] Preferably, the first administration of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 is separated from the first administration of the therapeutically effective amount of the platinum chemotherapy drug by 2 days.

[0043] In an optional embodiment, the first to fourth administrations of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are administered at intervals of 1 to 7 days.

[0044] In an optional embodiment, the administration cycle to the subject is 1 to 3 times, and the interval between each administration cycle is 5 to 7 days.

[0045] In an optional embodiment, the administration of the combination therapeutic pharmaceutical composition includes one or more of subcutaneous administration near the tumor and intratumoral injection.

[0046] In an optional embodiment, the combination therapeutic drug composition further comprises other tumor therapeutic drugs. Preferably, the other tumor therapeutic drugs are immunotherapeutic drugs. Further preferably, the immunotherapeutic drugs are PD-1 / PD-L1 inhibitors.

[0047] The present invention also aims to provide a drug kit or drug combination for treating tumors or cancers, comprising a therapeutically effective amount of a platinum chemotherapy drug, a therapeutically effective amount of a CpG oligonucleotide, and a therapeutically effective amount of R848;

[0048] Preferably, the platinum chemotherapy drug is selected from one or more of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin; preferably, the platinum is cisplatin; the therapeutically effective amount of CpG oligonucleotide (CpG ODN) is a class B CpG ODN or a class C CpG ODN;

[0049] More preferably, the class B CpG ODN is selected from one or more of ODN 1826, ODN 1018, and ODN 2006 / 7909;

[0050] More preferably, the C-class CpG ODN is selected from one or more of ODN M362, ODN 2395, and D-SL03;

[0051] The therapeutically effective amount of R848 is selected from one or more of water-soluble R848 and fat-soluble R848.

[0052] Optionally, instructions for use of the drug are also included.

[0053] Specifically, the therapeutically effective amount of the platinum chemotherapy drug, the therapeutically effective amount of the CpG oligonucleotide, and the therapeutically effective amount of R848 are packaged separately, or the therapeutically effective amount of the platinum chemotherapy drug is packaged separately, and the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are mixed and packaged;

[0054] Preferably, the therapeutically effective amount of the platinum chemotherapy drug, the therapeutically effective amount of the CpG oligonucleotide, and the therapeutically effective amount of R848 are sequentially administered to a subject in need thereof;

[0055] More preferably, the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are administered simultaneously, and the therapeutically effective amount of the platinum chemotherapy drug is administered before the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848;

[0056] More preferably, the therapeutically effective amount of the platinum chemotherapy drug is administered once, and the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are administered 1 to 5 times as one administration cycle;

[0057] The time interval between the first administration of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 and the first administration of the therapeutically effective amount of the platinum chemotherapy drug is 1 to 5 days;

[0058] Preferably, the first administration of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 is separated from the first administration of the therapeutically effective amount of the platinum chemotherapy drug by 2 days;

[0059] wherein the therapeutically effective amount of CpG oligonucleotide and the therapeutically effective amount of R848 are administered for the first to fifth times at intervals of 1 to 14 days;

[0060] The therapeutically effective amount of CpG oligonucleotide and the therapeutically effective amount of R848 are administered to the subject in a cycle of 1 to 3 times, with an interval of 5 to 7 days between each administration cycle.

[0061] Further preferably, the drug kit or drug combination further comprises other tumor therapeutic drugs, preferably, the other tumor therapeutic drugs are immunotherapeutic drugs, further preferably, the immunotherapeutic drugs are PD-1 / PD-L1 inhibitors.

[0062] A fourth object of the present invention is to provide use of the above-mentioned composition, kit or drug combination in the preparation of a drug for treating tumors or cancer.

[0063] In an optional embodiment, the tumor or cancer is selected from breast cancer, melanoma, liver cancer, basal cell carcinoma, cutaneous squamous cell carcinoma, cutaneous T-cell lymphoma, and colorectal cancer; preferably, the tumor or cancer is selected from breast cancer and melanoma.

[0064] A fifth object of the present invention is to provide a method for treating tumors or cancers, comprising administering to a subject a therapeutically effective amount of a platinum chemotherapy drug, a therapeutically effective amount of a CpG oligonucleotide, and a therapeutically effective amount of R848;

[0065] Preferably, the platinum chemotherapy drug is selected from one or more of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin; preferably, the platinum is cisplatin; the therapeutically effective amount of CpG oligonucleotide (CpG ODN) is a class B CpG ODN or a class C CpG ODN;

[0066] More preferably, the class B CpG ODN is selected from one or more of ODN 1826, ODN 1018, and ODN 2006 / 7909;

[0067] More preferably, the C-class CpG ODN is selected from one or more of ODN M362, ODN 2395, and D-SL03;

[0068] The therapeutically effective amount of R848 is selected from one or more of water-soluble R848 and fat-soluble R848.

[0069] In an optional embodiment, the therapeutically effective amount of the platinum chemotherapy drug is administered at a dosage of 2 μg / kg to 4000 μg / kg;

[0070] The therapeutically effective amount of the CpG oligonucleotide is administered in a dosage of 0.001 mg / time to 32 mg / time;

[0071] The therapeutically effective amount of R848 is administered in a dosage of 0.001 mg / time to 5 mg / time.

[0072] In an optional embodiment, the therapeutically effective amount of the platinum chemotherapy drug is administered in a dosage of 2 μg / kg to 20 μg / kg;

[0073] The therapeutically effective amount of the CpG oligonucleotide is administered in a dosage of 0.001 mg / time to 32 mg / time;

[0074] The therapeutically effective amount of R848 is administered in a dosage of 0.001 mg / time to 1 mg / time.

[0075] In a preferred embodiment, the therapeutically effective amount of the platinum chemotherapy drug is administered in a dosage of 2 μg / kg to 400 μg / kg;

[0076] The therapeutically effective amount of the CpG oligonucleotide is administered in a dosage of 0.05 mg / time to 2 mg / time;

[0077] The therapeutically effective amount of R848 is administered in a dosage of 0.01 mg / time to 1 mg / time.

[0078] In an optional embodiment, the therapeutically effective amount of the platinum chemotherapy drug, the therapeutically effective amount of the CpG oligonucleotide, and the therapeutically effective amount of R848 are packaged separately, or the therapeutically effective amount of the platinum chemotherapy drug is packaged separately, and the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are mixed and packaged;

[0079] In an optional embodiment, the therapeutically effective amount of a platinum chemotherapy drug, the therapeutically effective amount of a CpG oligonucleotide, and the therapeutically effective amount of R848 are sequentially administered to a subject in need thereof;

[0080] More preferably, the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are administered simultaneously, and the therapeutically effective amount of the platinum chemotherapy drug is administered before the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848;

[0081] More preferably, the therapeutically effective amount of the platinum chemotherapy drug is administered once, and the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are administered 1 to 5 times as one administration cycle;

[0082] The time interval between the first administration of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 and the first administration of the therapeutically effective amount of the platinum chemotherapy drug is 1 to 5 days;

[0083] Preferably, the first administration of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 is separated from the first administration of the therapeutically effective amount of the platinum chemotherapy drug by 2 days;

[0084] wherein the therapeutically effective amount of CpG oligonucleotide and the therapeutically effective amount of R848 are administered for the first to fifth times at intervals of 1 to 14 days;

[0085] The therapeutically effective amount of CpG oligonucleotide and the therapeutically effective amount of R848 are administered to the subject in a cycle of 1 to 3 times, with an interval of 5 to 7 days between each administration cycle.

[0086] In an optional embodiment, the tumor or cancer is selected from breast cancer, melanoma, liver cancer, basal cell carcinoma, cutaneous squamous cell carcinoma, cutaneous T-cell lymphoma, and colorectal cancer; preferably, the tumor or cancer is selected from breast cancer and melanoma.

[0087] Compared with the prior art, the present invention has the following beneficial effects:

[0088] The present invention provides a combined therapeutic pharmaceutical composition and a combined treatment method for tumor treatment, wherein the combined therapeutic pharmaceutical composition is a low-dose platinum chemotherapy drug combined with CpG and R848; the combined treatment method is to use low-dose platinum chemotherapy drugs, CpG and R848 at a specific time. The mechanism of action of the combined therapeutic pharmaceutical composition and combined treatment method for tumor treatment provided by the present invention is: first, a low-dose chemotherapy drug is used to kill the tumor to release tumor neoantigens, allowing the body's immune system to autonomously recognize and screen the neoantigens, and then a Toll-like receptor activator is used to stimulate innate and adaptive anti-tumor immune responses, that is, CpG oligonucleotides and R848 immune activators are used to induce and promote the body to produce tumor neoantigen-specific immune responses, enhance and strengthen the anti-tumor function of T cells, thereby achieving a broad-spectrum inhibition of tumor growth.

[0089] Specifically, the combination therapy of the present invention has the following advantages compared with other existing therapies: (1) significantly reducing the dosage of chemotherapy drugs, reducing the side effects caused by chemotherapy in patients, and improving compliance; (2) not being restricted by tumor types and the continuous mutation of tumor antigens, low-dose chemotherapy drugs release tumor antigens, and TLR activators activate the body's inherent anti-tumor immunity, responding to tumor mutations at any time; (3) having a wide range of applications, it can be applied to tumor patients who do not have surgical indications, is also suitable for neoadjuvant therapy to increase the patient's surgical treatment opportunities, can also be applied to patients with advanced malignant tumors who are ineffective with other treatment methods, and can also be used for postoperative adjuvant therapy. Its scope of application and prospects may be superior to existing passive immunotherapy or immune checkpoint blockade therapy products or methods. (4) It is low-priced, significantly reducing the financial burden on patients and their families, and alleviating the burden on national medical insurance. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific implementation or the description of the prior art.

[0091] FIG1 is a graph showing the growth curve of a 4T1 tumor provided in Example 1 of the present invention;

[0092] FIG2 is a picture of the exfoliated solid tumors in each group provided in Example 1 of the present invention;

[0093] FIG3 is a graph showing the tumor weight results of each group provided in Example 1 of the present invention;

[0094] FIG4 is a diagram showing the phenotype detection results of infiltrating lymphocytes in tumor tissues provided in Example 2 of the present invention;

[0095] FIG5 is a diagram showing the results of immune cell phenotype detection in draining lymph nodes provided in Example 2 of the present invention;

[0096] FIG6 is a graph showing the effect of different administration times on 4T1 tumor growth curves provided in Example 3 of the present invention;

[0097] FIG7 is a diagram showing the tumor inoculation and administration sites and a tumor growth curve result diagram provided in Example 4 of the present invention;

[0098] FIG8 is a graph showing the growth curve of B16 tumors provided in Example 5 of the present invention;

[0099] FIG9 is a graph showing the results of in vitro stimulation of mouse splenocytes by CpG and R848 from different sources to secrete TNF-α cytokines provided in Example 6 of the present invention;

[0100] FIG10 is a graph showing the tumor growth inhibition curves of CpG and R848 from different sources provided in Example 6 of the present invention;

[0101] FIG11 is a graph showing the weight of mice provided in Example 7 of the present invention;

[0102] FIG12 is a graph showing the volume growth curve of 4T1 tumors provided in Example 7 of the present invention;

[0103] FIG13 is a graph showing the tumor weight results of each group provided in Example 7 of the present invention;

[0104] FIG14 is a picture of the exfoliated solid tumors in each group provided in Example 7 of the present invention;

[0105] Figure 15 shows the expression of Ly6C monocytes and CD4 in draining lymph nodes provided in Example 8 of the present invention. + and CD8 +The expression results of IFNγ, TNFα, perforin and GzmB in T cells;

[0106] FIG16 shows the secretion levels of IFNγ, IFNα, and TNFα in the serum of each group of mice provided in Example 8 of the present invention;

[0107] FIG17 shows the quantitative results of T cells and B cells infiltrating in tumor tissues and the immunofluorescence labeling results provided in Example 8 of the present invention;

[0108] FIG18 is a graph showing the volume growth curves of B16 tumors in each group provided in Example 9 of the present invention;

[0109] FIG19 is a picture of the exfoliated solid tumors in each group provided in Example 9 of the present invention;

[0110] FIG20 is a graph showing the tumor weight results of each group provided in Example 9 of the present invention;

[0111] FIG21 is a graph showing the weight results of mice in each group provided in Example 9 of the present invention;

[0112] FIG22 is a graph showing the weights of important organs of mice in each group according to Example 9 of the present invention;

[0113] FIG23 is a flowchart of animal drug administration provided in Example 10 of the present invention;

[0114] FIG24 is a graph showing changes in mouse body weight during drug administration according to Example 10 of the present invention;

[0115] FIG25 is a curve of mouse tumor volume growth during drug administration provided in Example 10 of the present invention;

[0116] FIG26 is a survival curve of tumor-bearing mice during drug administration provided in Example 10 of the present invention;

[0117] FIG27 is a graph showing changes in mouse body weight during drug administration according to Example 11 of the present invention;

[0118] FIG28 is a curve of mouse tumor volume growth during drug administration provided in Example 11 of the present invention;

[0119] FIG29 is a survival curve of tumor-bearing mice during drug administration provided in Example 11 of the present invention;

[0120] FIG30 is a graph showing changes in mouse body weight during drug administration according to Example 12 of the present invention;

[0121] FIG31 is a curve of mouse tumor volume growth during drug administration provided in Example 12 of the present invention;

[0122] FIG32 is a survival curve of tumor-bearing mice during drug administration provided in Example 12 of the present invention;

[0123] FIG33 is a graph showing changes in mouse body weight during drug administration according to Example 13 of the present invention;

[0124] FIG34 is a curve of mouse tumor volume growth during drug administration provided in Example 13 of the present invention;

[0125] FIG35 is a survival curve of tumor-bearing mice during drug administration provided in Example 13 of the present invention;

[0126] FIG36 is a graph showing the 4T1 tumor volume growth curve results provided in Example 14 of the present invention. DETAILED DESCRIPTION

[0127] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0128] The technical solutions and beneficial effects of the present invention are further described below in conjunction with preferred embodiments.

[0129] Example 1. Tumor inhibition effect of low-dose cisplatin combined with CpG and R848 adjuvants

[0130] Balb / c mice were randomly divided into groups. The hair on the right back of each mouse was shaved near the buttocks using a mouse razor. 4T1 breast cancer cell suspension (3×10 5 / ) to construct the tumor model. When the tumor grows to an appropriate size, about 10mm 2 , which was formed about 3 days after inoculation, and the mice were administered with PBS control group, cisplatin (CDDP) control group, CpG+R848 (CR) adjuvant alone administration group, and CDDP combined with CR adjuvant administration group.

[0131] Specific mouse dosing groups: (1) CDDP 2μg (cisplatin 2μg / kg), (2) CDDP 5μg (cisplatin 5μg / kg), (3) CDDP 2μg + CR 10μg (cisplatin 2μg / kg, CpG 10μg / mouse, R848 10μg / mouse), (4) CDDP 5μg + CR 10μg (cisplatin 5μg / kg, CpG 10μg / mouse, R848 10μg / mouse), (5) CDDP 2μg + CR 50μg (cisplatin 2μg / kg, CpG 50μg / mouse, R848 50μg / mouse), (6) CR 10μg (CpG 10μg / mouse, R848 10μg / mouse), (7) CR 50μg (CpG 50 μg / mouse, R848 50 μg / mouse), (8) PBS negative control group. The full name of CpG is CpG oligonucleotide (CpG ODN).

[0132] Drug preparation: CDDP was dissolved in PBS and prepared into the concentration corresponding to the required dosage per mouse in a volume of 100ul. R848 was dissolved in a dedicated aqueous solvent into a 10mg / ml stock solution (Stock Solution). For each administration, 0.1ml of the Stock Solution was taken and the volume was adjusted to 1ml to prepare a 1mg / ml solution. The CpG used in this experiment was ODN 1826 (CpG1826), which was prepared into 1mg / ml in PBS before each administration. When administering, the 1mg / ml CpG1826 solution and the R848 solution were mixed in equal volumes according to the required amount to form different formulated "CR" mixed adjuvants. Each mouse was given 100ul of the mixed "CR" mixed adjuvant. CDDP is a product of Dalian Meilun Biotechnology Co., Ltd., product catalog number MB1055; R848 is a product of InvivoGen, product catalog number tlrl-r848-5; CpG1826 was purchased from Zixi Biotechnology Co., Ltd., product catalog number NS-004984-001.

[0133] Dosing schedule: The day of tumor implantation was designated Day 0. On Day 3, the CDDP group and the CR adjuvant and CDDP combination group were each administered 100 μl of CDDP. The PBS and CR groups were each administered 100 μl of PBS. The CR adjuvant group and the CR adjuvant and CDDP combination group were each administered 100 μl of the CR mixture on Days 5, 12, and 19, respectively. The PBS and CDDP control groups were administered 100 μl of PBS at the corresponding times. Administration was by subcutaneous injection adjacent to the tumor. Mice were sacrificed four days after the last dose.

[0134] Tumor size measurement: Tumor volume was measured twice a week using a vernier caliper. The long and short diameters of the tumor were measured. The tumor volume was calculated using the following formula: tumor volume = 0.5 × long diameter × short diameter 2 (mm 3 The formula for calculating tumor area is: tumor size = long diameter × short diameter (mm 2 ).

[0135] Tumor weighing: At the end of the experiment, the mouse tumor tissue was removed, weighed and recorded using an analytical balance, and the tumor was photographed and recorded.

[0136] Conclusion: The tumor size growth curves of mice in each drug-treated group are shown in Figure 1. As the number of days increased, it was found that the tumor in the PBS control group grew rapidly, while the tumor in the fifth group of mice treated with low-dose CDDP combined with high-dose CR adjuvant (CDDP 2μg+CR 50μg group) grew the slowest, and cell growth and proliferation were significantly inhibited, which had the best effect in inhibiting tumor growth compared with other groups.

[0137] The solid tumors detached from each group of mice are shown in Figure 2, and the corresponding tumor weight results are shown in Figure 3. It was found that the tumors grown in the high-dose CDDP combined with low-dose CR adjuvant group (CDDP 5μg+CR 10μg group), the low-dose CDDP combined with high-dose CR adjuvant group (CDDP 2μg+CR 50μg group), and the high-dose CR adjuvant alone group (CR 50μg group) were the lightest compared with the other groups.

[0138] Example 2. Flow cytometric analysis of immune cell phenotypes in tumor tissue and draining lymph nodes

[0139] The experimental groups were the same as those in Example 1, with the addition of a group of naive mice that were not inoculated with tumors and did not receive drugs. The construction of the 4T1 breast cancer cell tumor model, drug configuration, and administration schedule were the same as those in Example 1. Mice were sacrificed 7 days after the last administration, and tumors and lymph nodes were removed. Flow cytometry was used to detect the number of CD8 TILs in the tumor tissue. + T cells and CD4 + T cells.

[0140] Tumor tissue samples: Mice were sacrificed by cervical dislocation, and tumors were removed and weighed. Tumor tissue was minced into small pieces, and digested with 5.95 ml of RPMI 1640 medium containing 0.48 mg of collagenase solution and 5 μl of 1000 U / ml DNase I. The cells were then incubated at 37°C for 45 min. After digestion, 3 ml of complete medium was added to terminate the digestion process. The filtered medium was added to 10 ml in a 15 ml centrifuge tube and centrifuged at 400 g for 5 min. The cells were then resuspended and stained with flow cytometry staining mix and stained with Fixable Viability dye for 15 min. The cells were then washed and stained with anti-mouse CD3, CD8a, CD223 (LAG3), CD366 (TIM3), and CD279 (PD-1) antibodies for 15 min in the dark. The cells were then terminated with 150 μl of PBS. After centrifugation, the supernatant was discarded, the cells were resuspended, and analyzed by flow cytometry. The samples were detected by LSRFortessa, and the acquired flow data were analyzed on Flowjo software.

[0141] Lymph node samples: Mice were sacrificed by cervical dislocation, and lymph nodes were removed and placed in a 1.5 ml EP centrifuge tube. 300 μl of PBS was added and the sample was ground with a pestle. After filtration, the sample was centrifuged. Lymph node samples were directly resuspended in PBS. Fixable Viability dye was used for 15 minutes, followed by washing and staining with anti-mouse CD3, CD4, CD223 (LAG3), and CD279 (PD-1) antibodies for 15 minutes at room temperature in the dark. After washing, cells were permeabilized with fixation / permeabilization buffer for 1 hour and intracellularly stained with anti-mouse Foxp3 for 60 minutes at room temperature. After centrifugation, the sample was resuspended in PBS and analyzed. Samples were analyzed using an LSRFortessa, and acquired flow cytometric data were analyzed using FlowJo software.

[0142] Conclusion: The results of tumor tissue sample detection are shown in Figure 4. Figure 4A shows that the CD8 + The number of T cells was the highest, significantly better than that of other groups, indicating that this group had a better effect in activating CD8 + T cell immune response has excellent effect, inducing CD8 + T cells play an immune killing role, thereby inhibiting tumor growth. LAG3, TIM3, PD-1, and Foxp3 are related inhibitory molecules that induce T cell exhaustion. The results of Figure 4B-4C show that the expression of LAG3 in tumor tissues of the low-dose CDDP combined with high-dose CR adjuvant group (CDDP 2μg+CR 50μg group) is significantly higher than that of the control group (P<0.05).+ CD8 + T cells, TIM3 + CD8 + T cells, PD-1 + CD8 + The T cells were significantly lower than those in other groups, indicating that this group could inhibit CD8 + T cell depletion, making more CD8 + T cells perform effector functions to inhibit tumor growth.

[0143] The results of lymph node sample detection are shown in Figure 5. The results in Figures 5A-5C show that the expression of LAG3 in lymph node tissues of the low-dose CDDP combined with high-dose CR adjuvant group (CDDP 2μg+CR 50μg group) was significantly higher than that in the control group (CDDP 2μg+CR 50μg group). + CD4 + T cells, PD-1 + CD4 + T cells, Foxp3 + CD4 + The T cells in this group were significantly lower than those in the other tumor-inoculated and drug-treated groups, and the levels in this group were comparable to those in the naive mice that were not inoculated with tumors and did not use drugs, indicating that the CD4 + The expression of T cell exhaustion-related inhibitory molecules and the number of Treg cells were the lowest, indicating that this group could prevent CD4 + T cell depletion, making more CD4 + T cells perform effector functions to inhibit tumor growth.

[0144] Based on the results of Examples 1-2, it can be reasonably inferred that the low-dose CDDP combined with high-dose CR adjuvant medication group (CDDP 2 μg+CR 50 μg group) has the best tumor treatment effect and is a regimen that can be used in subsequent clinical studies.

[0145] Example 3. Exploration of administration time for CDDP combined with CpG and R848 to inhibit tumor growth

[0146] The above experiments explored the optimal dosage range of CDDP combined with CpG and R848. This example mainly explored whether different administration times would affect the effect of inhibiting tumor growth. The construction of the 4T1 breast cancer cell tumor model, drug configuration, and tumor size measurement were the same as in Example 1. The experimental administration groups were: (1) PBS negative control group, (2) CR (CpG 10 μg / mouse, R848 10 μg / mouse), (3) CDDP (cisplatin 2 μg / kg), (4) CDDP + CR (administration time 1) (cisplatin 2 μg / kg, CpG 10 μg / mouse, R848 10 μg / mouse), (5) CDDP + CR (administration time 2) (cisplatin 2 μg / kg, CpG 10 μg / mouse, R848 10 μg / mouse).

[0147] Dosage time: The day of tumor implantation is counted as day 0, and the method and site of administration is subcutaneous injection near the tumor.

[0148] (1) PBS group: 100 μl of PBS was administered on the 5th, 12th, and 19th days respectively;

[0149] (2) CR group: 100 μl of CR mixed drug was administered on the 5th, 12th, and 19th days respectively;

[0150] (3) CDDP group: 100 μl of CDDP was administered on day 5, and only 100 μl of PBS was administered on days 12 and 19;

[0151] (4) CDDP+CR (administration time 1): CDDP 100ul and CR mixed drug 100ul were administered on the 5th day, and only CR mixed drug 100ul each was administered on the 12th day and the 19th day.

[0152] (5) CDDP+CR (administration time 2, the same as that in Example 1): 100 μl of CDDP was administered on the 3rd day, and 100 μl of the CR mixed drug was administered on the 5th, 12th, and 19th days.

[0153] Conclusion: The tumor size growth curve is shown in Figure 6. As the number of days increases, the tumor in the PBS control group grows rapidly. The tumor in the CDDP+CR (dosage time 2) administration group grows the slowest compared with the other groups and is significantly better than the CDDP+CR (dosage time 1) administration group; this shows that the "dosage time 2" administration method provided by the present invention has a better effect in inhibiting tumor growth.

[0154] Example 4. Inhibitory effect of CDDP combined with CpG and R848 on tumors distal to the drug delivery

[0155] The above experiments were set up with single-point tumor inoculation and the administration site was subcutaneous injection near the tumor. It was proved that CDDP combined with CpG and R848 had a significant inhibitory effect on tumors near the administration site. In order to observe whether the tumor inhibition effect of CDDP combined with CpG and R848 can have the same inhibitory effect on distal tumors, 3×10 5 4T1 tumor cells were inoculated only on the right side of the tumor, and CDDP, CpG, and R848 were administered sequentially (Figure 7A, two-point tumor inoculation, only on the right side); a single-point tumor inoculation group was also set up, in which 3×10 5 4T1 cells were injected subcutaneously into the right side of the tumor (Figure 7B, single-point tumor inoculation, right-side administration). The administration time and tumor measurement method were the same as those in Example 1. Specific experimental administration groups were: (1) PBS negative control group, (2) CDDP (cisplatin 2 μg / kg), (3) single-point tumor inoculation CDDP+CR (cisplatin 2 μg / kg, CpG 50 μg / mouse, R848 50 μg / mouse), (4) double-point tumor inoculation CDDP+CR (cisplatin 2 μg / kg, CpG 50 μg / mouse, R848 50 μg / mouse).

[0156] Conclusion: The experimental results are shown in Figure 7C. As the number of days increased, the tumor in the PBS control group grew rapidly, while the growth of the tumor in the single-point inoculation CDDP+CR administration group, the right tumor in the two-point inoculation CDDP+CR administration group, and the left tumor in the two-point inoculation CDDP+CR administration group was significantly inhibited. The tumor grew the slowest and the tumor growth curves almost overlapped. This shows that when the tumor is inoculated at two points and only the right side is administered, that is, when the drug is administered unilaterally, CDDP combined with CpG and R848 can not only inhibit the growth of the right tumor, but also significantly inhibit the growth of the left tumor, indicating that CDDP combined with CpG and R848 can significantly inhibit the growth of proximal tumors and also significantly inhibit the growth of distal tumors, indicating that the combined therapeutic composition and its combined treatment method have an inhibitory effect on systemic tumor lesions.

[0157] Example 5. Inhibitory effect of low-dose cisplatin combined with CpG and R848 adjuvant on melanoma

[0158] To further explore whether low-dose CDDP combined with CpG and R848 adjuvants has the same tumor inhibitory effect on melanoma, we established a mouse melanoma B16-F10 tumor model and used 3×10 5The tumor model was constructed with 100 tumor cells per mouse. Drug configuration, administration time and method, and tumor size measurement were the same as in Example 1. Experimental dosing groups: (1) PBS negative control group, (2) CDDP 2 μg (cisplatin 2 μg / kg), (3) CR 50 μg (CpG 50 μg / mouse, R848 50 μg / mouse), (4) CDDP 2 μg + CR 50 μg (cisplatin 2 μg / kg, CpG 50 μg / mouse, R848 50 μg / mouse).

[0159] Conclusion: The melanoma tumor size growth curve is shown in Figure 8. As the number of days increases, it is found that the tumor in the PBS control group grows rapidly, while the tumor in the mouse group 4, CDDP 2μg + CR 50μg, grows the slowest, and cell growth and proliferation are significantly inhibited, which is the best in inhibiting tumor growth compared with other groups. This shows that the low-dose cisplatin combined with CpG and R848 adjuvants provided by the present invention (CDDP 2μg + CR 50μg group) has a significant tumor inhibitory effect not only on breast cancer cells, but also on melanoma cells, suggesting that the low-dose cisplatin combined with CpG and R848 adjuvants provided by the present invention has a broad-spectrum effect of inhibiting tumor growth.

[0160] Example 6. Comparison of the effects of CpG or R848 from different sources

[0161] Since the CpG and R848 used in all the above examples are research-grade reagents, pharmaceutical-grade reagents are required to promote the clinical application of this invention. Therefore, we conducted experiments to compare whether the research-grade reagents CpG1826 and R848 (water-soluble) and the pharmaceutical reagents CpG1018 (ODN 1018) and R848 (fat-soluble) have the same effect. CpG1018 was purchased from Zixi Biotechnology Co., Ltd., catalog number NS-007772-001, and R848 (fat-soluble) was purchased from Hubei Weideli Chemical Technology Co., Ltd., catalog number 144875-48-9.

[0162] Drug preparation: The preparation and use of scientific-grade CpG1826 and R848 (water-soluble) were the same as in Example 1. The medicinal reagent R848 (lipid-soluble) was dissolved in DMSO to a 10 mg / ml stock solution. For each administration, 0.1 ml of the stock solution was taken and the volume was adjusted to 1 ml to prepare a 1 mg / ml solution. CpG1018 was prepared in PBS to 1 mg / ml before each administration.

[0163] 1. In vitro experiments

[0164] Three spleens from blank BABL / c mice were collected and placed in a 30mm cell culture dish with 1640 medium. After grinding, the cells were filtered through a copper mesh and centrifuged at 1500 rpm for 3 minutes. The supernatant was discarded and 2 ml of red blood cell lysis buffer was added. The cells were allowed to stand for 2 minutes and then terminated with 4 ml of 1640 + 10% FBS. The cells were centrifuged at 1500 rpm for 3 minutes. The supernatant was discarded and the cells were resuspended in 1640 + 10% FBS and counted. The cells were counted at 2x10 6 The cells were plated in 96-well cell culture plates with a culture volume of 200 μl per well. CpG and R848 at various doses were added. LPS was used as a positive control, and blank wells (NC) with only PBS were used as negative controls. After 18 to 20 hours of stimulation, the cell culture supernatant was collected and the TNF-a detection kit for mice (purchased from Xinbosheng Biotechnology Co., Ltd., catalog number EMC102a.96) was used to measure the TNF-a level every 10 min according to the manufacturer's instructions. 6 Cytokine expression in spleen cells of mice.

[0165] Conclusion: The experimental results are shown in Figure 9. Figure 9A shows that research-grade R848 (water-soluble) and pharmaceutical-grade R848 (lipid-soluble) stimulated BALB / c mouse splenocytes to produce comparable levels of TNF-α cytokines in vitro. Figure 9B also shows that research-grade CpG1826 and pharmaceutical-grade CpG1018 stimulated BALB / c mouse splenocytes to produce comparable levels of TNF-α cytokines in vitro. This indicates that changing the type and source of CpG and R848 had no significant effect on the levels of cytokine production by mouse splenocytes, demonstrating that pharmaceutical-grade CpG and R848 have the same efficacy as research-grade reagents.

[0166] 2. In vivo experiments

[0167] The construction of the 4T1 breast cancer cell tumor model, the administration time and method, and the tumor size measurement were the same as in Example 1. The experimental administration groups were: (1) PBS negative control group, (2) CDDP (cisplatin 2 μg / kg), (3) CDDP+CR (cisplatin 2 μg / kg, CpG1826 50 μg / mouse, R848 (water-soluble) 50 μg / mouse), and (4) CDDP+C'R' (cisplatin 2 μg / kg, CpG1018 50 μg / mouse, R848 (lipid-soluble) 50 μg / mouse).

[0168] Conclusion: The experimental results are shown in Figure 10. The tumor growth of mice in the CDDP+CR group and the CDDP+C'R' group was the slowest, and the cell growth and proliferation were significantly inhibited. The two groups had similar effects in inhibiting tumor growth, further indicating that pharmaceutical-grade CpG1018 and R848 (lipid-soluble) have the same tumor-inhibiting effect as scientific-grade reagents, laying the foundation for clinical application.

[0169] Example 7. Inhibitory effects of different formulations of cisplatin combined with CpG and R848 adjuvants on 4T1 breast cancer tumors

[0170] To further explore the tumor inhibition effect after the formula change, the experimental drug groups were as follows: 6 mice per group, (1) PBS negative control group, (2) CDDP (cisplatin 2 μg / kg), (3) CR (CpG1826 50 μg / mouse, water-soluble R848 50 μg / mouse), (4) CDDP+CR (cisplatin 2 μg / kg, CpG1826 50 μg / mouse, water-soluble R848 50 μg / mouse), (5) C'R' (CpG1018 50 μg / mouse, fat-soluble R848 50 μg / mouse), (6) CDDP+C'R' (cisplatin 2 μg / kg, CpG1018 50 μg / mouse, fat-soluble R848 50 μg / mouse), (7) CDDP+C'R (cisplatin 2 μg / kg, CpG1018 50 μg / mouse, water-soluble R848 50 μg / mouse), (8) CDDP+CR' (cisplatin 2 μg / kg, CpG1826 50 μg / mouse, lipid-soluble R848 50 μg / mouse). The construction of the 4T1 breast cancer cell tumor model, the dosing procedure, and tumor size measurement were the same as in Example 1. The configuration of CpG and R848 was the same as in Example 6.

[0171] 7.1 Mouse body weight measurement

[0172] After tumor inoculation, the weight of the mice was measured every 3 or 4 days and the changes in the injection site were observed. No swelling or ulceration was found at the injection site in each group of mice. In addition, in the combined drug administration group, as shown in Figure 11, the weight of the mice in each group was relatively stable, with a slight downward trend after each administration, and no continuous decline, and recovery after 3 days.

[0173] 7.2 Tumor volume measurement

[0174] After tumor inoculation, the tumor volume of mice was measured every 3 or 4 days. The tumor volume growth curves of mice in each drug-treated group are shown in Figure 12. As the number of days increased, it was found that the tumors in the PBS control group and the CDDP alone drug-treated group grew rapidly, while the growth and proliferation of tumor cells in the other combined drug-treated groups were significantly inhibited. All of them had good tumor inhibitory effects, among which the CDDP+CR group and CDDP+C'R group had the best tumor inhibitory effects.

[0175] 7.3 Tumor Weight Measurement and Mouse Survival Record

[0176] At the end of the experiment, tumor tissue was removed from the mice, weighed and recorded using an analytical balance, and photographed. The corresponding tumor weight results are shown in Figure 13. Tumors in the PBS control group and the CDDP alone group grew excessively large and heavy, while tumors in the other groups were significantly lighter than those in the two groups. The CDDP+CR group had the lightest tumor. The solid tumors removed from each group of mice are shown in Figure 14. One mouse in each of the C'R' and CDDP+C'R' groups died midway after drug administration. One mouse in each of the CR, CDDP+CR, and CDDP+C'R' groups experienced tumor remission after drug administration. Three mice in the CDDP+C'R group experienced tumor remission after drug administration, and no mice in the CDDP+C'R group died after drug administration.

[0177] Based on the experimental results of this example, and considering safety and tumor inhibition effects, the pharmaceutical composition of CDDP+C'R was selected as the preferred formulation, and the tumor adjuvant (C'R) composed of CpG1018 and water-soluble R848 was named CR108 for subsequent experiments.

[0178] Example 8 Study on the mechanism of the effect of cisplatin combined with CR108 adjuvant on immunity

[0179] 5×10 5 The number of tumor cells per mouse was 100 μg / kg, and a mouse breast cancer 4T1 tumor model was constructed. The drug administration process was the same as that in Example 7. The experimental drug administration groups were: 5 mice per group, (1) PBS negative control group, (2) CDDP (cisplatin 2 μg / kg), (3) CDDP + CR108 (cisplatin 2 μg / kg, CpG1018 50 μg / mouse, water-soluble R848 50 μg / mouse), (4) CR108 (CpG1018 50 μg / mouse, water-soluble R848 50 μg / mouse).

[0180] 8.1 Flow cytometry detection of immune cells

[0181] Fluorescently labeled antibodies used include: Biolegend anti-mouse CD4 (GK1.5), CD8a (53-6.7), CD11b (M1 / 70), and Ly6C (HK1.4); eBioscience anti-mouse perforin (eBioOMAK-D), CD3e (145-2C11), TNFα (MP6-XT22), anti-mouse IFNγ (XMG1.2), Granzyme B (NGZB), and Fixable Viability dye eFluor 780. Mice were sacrificed at the appropriate time after drug administration, and draining lymph nodes (dLNs) were harvested and prepared into a lymphocyte suspension. The resulting lymphocytes were washed once with PBS and stained with cell surface antibodies for 15 minutes at room temperature. For detection of intracellular antigens, cells were permeabilized with fixation / permeabilization buffer for 1 hour after cell surface antibody staining, followed by staining with intracellular antibodies for 1 hour at room temperature. After washing and resuspension, all stained samples were detected on LSRFortessa (BD Biosciences), and the acquired flow cytometry data were analyzed on FlowJo software.

[0182] Conclusion: On the third day after the first administration of CR108, the level of immune cell Ly6C monocytes in the draining lymph nodes of the CDDP+CR108 group was significantly increased compared with the other groups (Figure 15A), indicating that the CDDP+CR108 group has a better ability to enhance the immune system of tumor-bearing mice. IFNγ, TNFα, perforin and granzyme (GzmB) play an important role in the anti-tumor immune system. The pores formed by perforin allow granzymes to enter tumor cells and cause tumor cell apoptosis. As shown in Figures 15B-15G, experiments were conducted three days after the second administration of CR108, and it was found that IFNγ in the lymph node tissues of tumor-bearing mice in the CDDP+CR108 group was significantly increased. + CD4 + T cells, TNFα + CD4 + T cells, IFNγ + CD8 + T cells, TNFα + CD8 + T cells, perforin + CD8 + T cells and GzmB + CD8 + The percentage of T cells was significantly higher than that of other groups, indicating that the CDDP+CR108 administration group led to the increase of CD8 + and CD4 +The activation of T cells promotes a significant increase in the levels of IFNγ, TNFα, perforin and granzyme, thereby achieving excellent anti-tumor effects.

[0183] 8.2 ELISA method for cytokine detection

[0184] Furthermore, we also measured the secretion of IFNγ, IFNα, and TNFα in the serum of tumor-bearing mice using a multi-cytokine ELISA kit (Multi Sciences) according to the instructions. The concentrations of IFNγ and TNFα in the serum of tumor-bearing mice were measured 3 hours after the first CR108 administration and 4 hours after the second CR108 administration. As shown in Figures 16A-16C, the secretion levels of IFNγ, IFNα, and TNFα in the CDDP+CR108 treatment group were significantly higher than those in the other groups.

[0185] 8.3 Detection of T and B Cells Infiltrating Tumor Tissue by Flow Cytometry and Fluorescence Staining

[0186] CD4 infiltrating in tumor tissue + T, CD8 + T, B220 + B and PD1 + CXCR5 + Quantification of Tfh cells: The mouse tumors were collected on the 7th day after the third CR108 administration, and the tumor tissues were cut into small pieces. The small pieces of tumor tissue were incubated with type IV collagenase (Sigma, 80 μg / ml) and DNase I (Sigma) (50 U / ml) for 45 minutes. The tumor pieces were then mechanically dispersed and filtered through a 40 mm filter. Finally, flow antibody staining and flow cytometric analysis were performed; the flow staining antibodies used included CD45 (30-F11), CD4 (GK1.5), CD8a (53-6.7), B220 (RA3-6B2), CXCR5 (L138D7), and PD-1 (RMP1-30).

[0187] Immunofluorescence labeling of infiltrating T and B cells in tumor tissue: Tumors were collected from mice 7 days after the third dose of CR108. Tumor tissue was fixed in 4% paraformaldehyde (PFA) / PBS for over 24 hours and then embedded in paraffin. Modified tissue sections were sectioned on a paraffin microtome at a thickness of 4 μm. Primary antibodies used were: B220 (RA3-6B2, eBiosciences) and CD3 (GB111337, Servicebio). Secondary antibodies were: cyanin 3 Goat anti-rat IgG (Cy3, GB21302, Servicebio) and 488 Goat anti-rabbit IgG (GB25303, Servicebio). Sections were then incubated with TSA (Servicebio) in the dark for 10 minutes and then immersed in citrate fixation solution for microwave-assisted tissue fixation to enable multiple fluorescent staining. Cell nuclei were stained with DAPI (Sigma). Scanning was performed using a Pannoramic scanner (3D HITECH) and analysis was performed using Caseviewer.

[0188] Conclusion: We further detected the infiltration of immune cells in the tumor tissues of each group of mice treated with drugs. As shown in Figures 17A-17D, we found that compared with the other groups, the CD8 + T cells, CD4 + The number of T cells, Tfh cells, and B cells increased significantly, with the largest number of infiltrates. At the same time, the intuitive image of T cells and B cells infiltrating in tumor tissues detected by immunofluorescence labeling is shown in Figure 17E. A large number of T cells and B cells infiltrated in the tumor tissues of the CDDP+CR108 treatment group, further indicating that CDDP+CR108 can better enhance the immune response of tumor mice and promote immune cells to enter tumor tissues to exert anti-tumor effects.

[0189] Example 9. Inhibitory effect of cisplatin combined with CR108 adjuvant on mouse melanoma

[0190] This example further explored the effect of cisplatin combined with CR108 adjuvant on melanoma in mice. 5 The number of tumor cells / mouse was subcutaneously inoculated to construct a mouse melanoma B16-F10 tumor model. The tumor volume was about 20 mm on the 5th day after inoculation. 3 ~30mm 3 Perform medication administration.

[0191] Specific mouse dosing groups: (1) PBS negative control group, (2) CDDP Lo(cisplatin 2 μg / kg), (3) CR108 (CpG1018 50 μg / mouse, water-soluble R848 50 μg / mouse), (4) CDDP Lo +CR108 (cisplatin 2 μg / kg, CpG1018 50 μg / mouse, water-soluble R848 50 μg / mouse), (5) CDDP Lo +C' (cisplatin 2μg / kg, CpG1018 50μg / mouse), (6)CDDP Lo +R (cisplatin 2μg / kg, water-soluble R848 50μg / mouse), (7) CDDP Hi (Cisplatin 4 mg / kg, conventional cisplatin chemotherapy dose); except for group (7) where high-dose cisplatin was injected intraperitoneally, all other groups received subcutaneous injection near the tumor.

[0192] Dosage time: The day of tumor implantation is counted as day 0.

[0193] (1) PBS group: 100 μl of PBS was administered on the 5th, 7th, 14th, and 21st days respectively;

[0194] (2)CDDP Lo Group: 100ul of CDDP was given on day 5, and only 100ul of PBS was given on days 7, 14, and 21;

[0195] (3) CR108 group: 100 μl of PBS was administered on day 5, and 100 μl of CR108 mixed drug was administered on day 7, day 14, and day 21, respectively;

[0196] (4)CDDP Lo +CR108: 100ul of CDDP was administered on day 5, and 100ul of CR108 mixed drug was administered on days 7, 14, and 21.

[0197] (5)CDDP Lo +C': 100ul of CDDP was administered on day 5, and 100ul of CpG drug was administered on day 7, day 14, and day 21.

[0198] (6)CDDP Lo +R: 100 μl of CDDP was administered on day 5, and 100 μl of R848 was administered on days 7, 14, and 21.

[0199] (7)CDDP Hi Group: 100 μl of CDDP was administered on the 7th, 14th, and 21st days respectively;

[0200] 9.1 Tumor volume measurement

[0201] The tumor volume growth curves of the mice in each drug-treated group are shown in FIG18 . As the days increase, the CDDP Lo The growth and proliferation of tumor cells in the +CR108-treated group were significantly inhibited, with the tumor growing the slowest, the smallest in size, and basically no growth, which was significantly better than the other drug groups.

[0202] 9.2 Tumor Weight Measurement and Mouse Survival Record

[0203] On the 4th day after the last administration, the mice were killed, and the tumor tissues were removed and weighed using an analytical balance. The tumors were also photographed. The solid tumors removed from each group of mice are shown in Figure 19, and the corresponding tumor weight results are shown in Figure 20. Hi One mouse in the CDDP group died midway after administration. Lo The tumors in the +CR108 group were the lightest and smallest in size compared to those in the other groups. In addition, the tumor disappeared in one mouse after the drug administration, as shown by CDDP. Lo +CR108 has a significant tumor inhibition effect, which is significantly better than CpG, R848 alone combined with cisplatin (CDDP Lo +C' group, CDDP Lo +R group), and is superior to conventional cisplatin chemotherapy alone (CDDP Hi group) and has higher security.

[0204] 9.3 Mouse Body Weight Measurement

[0205] The results of mouse weight are shown in Figure 21. There was no significant change in the weight of each group. The overall weight data remained stable and increased slightly. There was no statistical difference in the weight of mice in each group at the end of the experiment, indicating that the tumor adjuvant CR108 has good safety.

[0206] 9.4 Measurement of the Weight of Important Organs in Mice

[0207] At the end of the experiment, the liver, lung, spleen, and lymph nodes of the mice were removed (the left side L is the lymph node on the side without tumor inoculation, and the right side R is the lymph node on the side with tumor inoculation), and the weights were weighed and recorded using an analytical balance. The weight measurement results of the mouse organs are shown in Figure 22. Figures 22A-D show that CDDP Lo The weight of the liver, lung, spleen, and lymph nodes of the mice in the C group increased to a certain extent, which was higher than that in the other groups. This indicates that when CpG is used alone in combination with cisplatin, it may cause excessive immune response in various organs, resulting in excessive swelling and weight increase. When the combination of R848+CpG and cisplatin (CDDP) is used in combination with CpG, the weight of the liver, lung, spleen, and lymph nodes of the mice in the C group increased to a certain extent, which was higher than that in the other groups. Lo+CR108 group) can reduce the excessive increase in liver, lung, spleen and lymph node weight caused by the use of CpG alone and cisplatin, while increasing the immune response of draining lymph nodes, with better safety and the best tumor inhibition effect.

[0208] Example 10 Study on the inhibitory effect and treatment regimen of cisplatin combined with CR108 adjuvant intratumoral injection on large-volume melanoma in mice

[0209] In all the above examples, the combination of micro-dose cisplatin and tumor adjuvant (CpG+R848) was verified to be effective in the treatment of small tumors (20-30 mm) in mouse breast cancer model and melanoma model. 3 ) not only has the effect of "reducing toxicity and increasing efficacy" in tumor treatment, but also tumor adjuvant therapy can stimulate the body's inherent anti-tumor immune effect without the need for tumor antigen immunity, while obtaining a stable tumor adjuvant formula. However, whether the treatment plan based on small tumors is applicable to large tumors (200-300mm) 3 Considering the actual situation of using intratumoral injection for tumor treatment in clinic, this example explores the effect of "low-dose chemotherapy drugs + tumor adjuvant" on tumors growing to a larger size (average of about 200mm) through different intratumoral injection procedures. 3 ) and the optimal administration procedure.

[0210] 3×10 5 The mouse melanoma B16-F10 tumor model was constructed with the number of tumor cells per mouse. The tumors grew to an appropriate size, with an average size of about 200 mm. 3 According to the tumor size and body weight, appropriate mice were selected and medicated according to the groups. The dosage of each drug was 2μg / kg CDDP, 50μg / mouse CpG1018, and 50μg / mouse water-soluble R848. Specific groups: G1 group: normal saline control group (NS), medicated once a week; G2 group: first intratumoral injection of CDDP, then intratumoral injection of CR108 two days later, once a week; G3 group: first intratumoral injection of CDDP in each dosing cycle, then intratumoral injection of CR108 two days later, once every other day, four times a cycle, and repeat the next dosing after one week of discontinuation; G4 group: CDDP and CR108 were mixed and injected into the tumor at the same time, and administered twice a week. The specific dosing flow chart is shown in Figure 23. After the dosing was completed according to the flow chart, the drug was discontinued. During this period, the tumor size and physiological state of the mice were observed. When the tumor volume of each mouse exceeded 2000mm for the first time, 3 , or when the animal's weight was less than 80% of its weight before the first administration, it was considered ethical death and the animal was euthanized, and the remaining mice continued to be observed.

[0211] 10.1 Mouse Body Weight Measurement

[0212] Before and during dosing, we continuously observed and measured the mice's weights at the cage side. Routine cage-side observations revealed that most mice experienced transient decreased activity, ruffled hair, and weight loss on the second day after administration of the tumor adjuvant, but all returned to normal by the third day. The mouse weight results are shown in Figure 24. The weights of mice in each group changed little from the start to the end of the experiment, indicating that the combination of micro-dose cisplatin and CR108 has good safety. All mice in Group G1 (normal saline control group) were ethically euthanized on Day 28. The experiment ended when more than half of the mice in all groups, except Group G3, were euthanized on Day 32.

[0213] 10.2 Tumor Volume Measurement

[0214] The tumor volume growth curves of the mice in each drug-treated group are shown in Figure 25. When the experiment was carried out to Day 28, all the mice in the G1 saline control group had tumor volumes exceeding 2000 mm 3 At the time of ethical death, the mean tumor volume was 2581.9±353.3. The mean tumor volume (mean±SEM) for the three dosing schedules and their T-test significance relative to the G1 control group were as follows: the mean tumor volume for the G2 group was 1490±363.3, p=0.0621; the mean tumor volume for the G3 group was 996.9±286.4, p=0.0064; and the mean tumor volume for the G4 group was 1654.4±347.9, p=0.0984. The results showed that when the tumor grew normally to the ethical limit, only the dosing schedule used in the G3 group had a significantly superior tumor inhibition effect than the saline control group (G1).

[0215] 10.3 Survival curves of tumor-bearing mice during treatment

[0216] As shown in Figure 26, all mice in the saline control group died on day 28. By the end of the experiment, the median survival of each group was 28 days for the saline control group, 33 days for the G2 group, 32 days for the G4 group, and 38 days for the G3 group. These data indicate that the G3 dosing schedule significantly prolonged the survival of mice bearing large melanoma tumors.

[0217] In this example, we preliminarily determined the intratumoral drug administration process G3 in a large-volume tumor model. We will continue to explore the use of the G3 group's drug administration process and increase the dose of cisplatin to observe whether it can improve the tumor inhibition effect.

[0218] Example 11 Study on the treatment of large-volume melanoma in mice by intratumoral injection of CR108 adjuvant combined with different doses of cisplatin

[0219] Example 10 The study determined the administration procedure of tumor adjuvant combined with cisplatin for the treatment of large melanoma. This example aims to optimize the cisplatin dosage in order to obtain a more significant tumor inhibition effect.

[0220] Experimental groups: (1) saline control group, the administration procedure was the same as that of Example 10G1 group; (2) CDDP Hi +CR108 (cisplatin dose 0.4 mg / kg), administration procedure was the same as that of group 10G2 in Example 1 (qw); (3) CDDP Lo +CR108 (cisplatin dose 2 μg / kg), administration procedure was the same as that of group 10G3 in Example 1 (qod); (4) CDDP Hi + CR108 (cisplatin 0.4 mg / kg), the administration procedure was the same as that of Group G3 in Example 10 (qod); the dose of CR108 used in each group was 50 μg / mouse of CpG1018 and 50 μg / mouse of water-soluble R848.

[0221] 11.1 Mouse Body Weight Measurement

[0222] The results of mouse weight are shown in Figure 27. Before and during the administration, the mice were observed and weighed continuously. Lo Except for one mouse that received +CR108(qod.), most mice were in good condition after administration. Only on the second day after each administration of the tumor adjuvant did the mice show a transient decrease in activity, ruffling of hair, and weight loss, but all returned to normal on the third day. The weight changes of mice in each group were small from the beginning to the end of the experiment, especially in the fourth group (CDDP Hi The body weights of the mice treated with cisplatin + CR108 (qod.) remained stable after administration, indicating that the 0.4 mg / kg cisplatin combined with CR108 dosage had good safety.

[0223] 11.2 Tumor Volume Measurement

[0224] The results are shown in Figure 28. When the experiment was carried out to Day 33, except for one tumor that was eaten by mice in the normal saline control group, the rest of the mice had tumors exceeding 2000 mm. 3 Ethical Death. Group (2) CDDP Hi Although the +CR108 (qw.) group also increased the dose of cisplatin, it was better than the CDDP of group (3) which increased the frequency of CR108 administration. Lo +CR108 (qod.) and Group (4) CDDP HiThe tumor inhibition effect of the two groups + CR108 (qod.) was still poor, which once again proved that increasing the frequency of CR108 administration (G3) can improve the tumor inhibition effect. Group (3) CDDP with trace cisplatin Lo +CR (qod.) had a certain inhibitory effect on tumor growth, which was basically consistent with the results of the administration procedure of Example 10 (Figure 25); while the CDDP group (4) with increased cisplatin dose Hi +CR (qod.) had the most significant inhibitory effect on melanoma, with virtually no tumor growth. This suggests that, at the same dosing frequency, appropriately increasing the dose of low-dose cisplatin can significantly improve the tumor-suppressing effect.

[0225] 11.3 Survival curves of tumor-bearing mice during treatment

[0226] As shown in Figure 29, all mice in the saline control group died on the 33rd day; the median survival time of each group at the end of the experiment was: 31 days for the saline control group, 31 days for the CDDP group, and 31 days for the CDDP group. Hi +CR108 (qw.) group: 37 days, CDDP Lo +CR108 (qod.) is 38 days; and CDDP Hi The +CR108(qod.) group had not yet reached the median survival time by Day 55 of long-term observation after two rounds of dosing, demonstrating the best survival extension in tumor-bearing mice. These results suggest that, under the same dosing schedule, appropriately increasing the cisplatin dose can significantly prolong the survival of mice bearing large melanoma tumors.

[0227] Example 12 Comparative study of intratumoral injection of CR108 adjuvant combined with subdose cisplatin and conventional therapeutic dose cisplatin in the treatment of large-volume melanoma in mice

[0228] This example mainly explores the difference between the dosing schedule and optimized cisplatin dose screened by Examples 10 and 11 and the conventional therapeutic dose of cisplatin.

[0229] When the tumor grows to an appropriate size, the average size is about 200mm 3 The mice were dosed according to the experimental groups: (1) normal saline control group, the dosing procedure was the same as that of Example 10G1 group; (2) CDDP (cisplatin 4 mg / kg, conventional cisplatin chemotherapy dose), the dosing procedure was once a week; (3) CR108, the dosing procedure was the same as that of Example 10G3 group, and only normal saline was injected at the time of cisplatin injection, without cisplatin injection; (4) CDDP+CR108 (cisplatin 0.4 mg / kg), the dosing procedure was the same as that of Example 10G3 group; the dose of CR108 used in each group was CpG1018 50 μg / mouse and water-soluble R848 50 μg / mouse.

[0230] 12.1 Mouse Body Weight Measurement

[0231] Mice were observed and weighed continuously at the cage side before and during dosing. Routine cage-side observation revealed that all mice were generally in good condition after dosing. Only on the second day after each administration of the tumor adjuvant did mice experience a transient decrease in activity, ruffling of hair, and weight loss, which returned to normal by the third day. With the exception of the saline control group, which experienced a slight weight gain due to rapid tumor growth, the weight of mice in the remaining dosing groups remained relatively stable from the start to the end of the experiment, further demonstrating the good safety of intratumoral injection of 0.4 mg / kg cisplatin combined with CR108 adjuvant. By Day 28, all mice in the saline group had died ethically, and the remaining mice were monitored until the end of dosing. Weight data for this period are shown in Figure 30.

[0232] 12.2 Tumor Volume Measurement

[0233] The results are shown in Figure 31. When the experiment was carried out to Day 28, the mice in the normal saline control group had tumor volumes exceeding 2000 mm. 3 Ethical death. In the control group of group (2) treated with 4 mg / kg CDDP conventional treatment, the tumor volume was significantly lower than that of the normal saline control group on Day 28, showing a certain tumor inhibition effect. However, on Day 36, all mice in this group had tumors exceeding 2000 mm 3 The tumor volumes of the (3rd) CR108-only treatment group and the (4th) CDDP combined with CR108-treatment group also showed a very significant tumor inhibition effect compared with the saline control group, and the CDDP+CR108-treatment group had a significantly better tumor inhibition effect than the CR108-only treatment group.

[0234] 12.3 Survival Curves of Tumor-Bearing Mice During Treatment

[0235] As shown in Figure 32, all mice in the saline control group died on day 28. By the end of the experiment, the median survival of each group was 26 days for the saline control group, 36 days for the group receiving 4 mg / kg of conventional cisplatin-CDDP, and 42 days for the group receiving CR108 alone. However, the median survival of the group receiving 0.4 mg / kg of CDDP combined with CR108 (CDDP+CR108) remained below the 45th day of long-term observation after two rounds of dosing, and the survival rate remained at 90%. The data demonstrate that the CDDP+CR108 treatment group significantly prolonged the survival of mice bearing large melanoma tumors compared to both the CR108-alone treatment group and the conventional cisplatin-CDDP chemotherapy group, demonstrating a significant tumor-suppressing effect.

[0236] Example 13 Study on the Tumor Inhibitory Effect of Combinations of Different Doses of CpG1018 and a Fixed Dose of R848

[0237] Through a series of experiments, we have determined that intratumoral drug administration for large tumor volume (tumor volume of about 200 3 ~300mm 3 ) Cisplatin dosage and administration schedule for mouse melanoma. Based on our previous demonstration that CpG1018 plays a primary role in the overall tumor inhibition process (results shown in Figures 19 and 20 ), this example further explored the inhibitory effect and safety of CR108, composed of different doses of CpG1018, on a large melanoma tumor model.

[0238] When the tumor grows to an appropriate size, the average size is about 200mm 3 The mice were dosed according to the experimental groups: (1) normal saline control group, the dosing procedure was the same as that of group G1 in Example 10; (2) CDDP + C50R108 (CpG1018 50 μg / mouse, water-soluble R848 50 μg / mouse); (3) CDDP + C100R108 (CpG1018 100 μg / mouse, water-soluble R848 50 μg / mouse); (4) CDDP + C200R108 (CpG1018 200 μg / mouse, water-soluble R848 50 μg / mouse), the dose of CDDP used in each group was 0.4 mg / kg, and the dosing procedures of groups (2), (3), and (4) were the same as those of group G3 in Example 10.

[0239] 13.1 Mouse Body Weight Measurement

[0240] The mice were observed and weighed before and during the administration. During routine cage observation, we found that all mice were in good condition after administration. Only on the second day after each administration of the tumor adjuvant, the mice showed a transient decrease in activity, ruffling of hair, and weight loss, but all returned to normal on the third day. The weight of mice in each group changed little from the beginning to the end of the experiment, especially in group (4) (CDDP+C 200 R108). This indicates that increasing the dose of CpG1018 within a certain range has a good safety profile. By Day 33, all patients in the saline group had died. Body weight data during this period are shown in Figure 33.

[0241] 13.2 Tumor Volume Measurement

[0242] The results are shown in Figure 34. When the experiment was carried out to Day 31, the tumor volume of the mice in the normal saline control group had exceeded 2000mm. 3Or it has begun to grow rapidly to the ethical death threshold. The tumor inhibition effect of CR108 adjuvant composed of different doses of CpG1018 is basically the same. Compared with the normal saline control group mice, groups (2), (3), and (4) had significant tumor inhibition effects on Day 31, and the inhibition rates on Day 31 were 78.51%, 73.17%, and 88.39%, respectively. After one round of administration, there was no significant difference between the CpG administration groups at different doses on Day 31. After Day 31, it can be clearly seen that the anti-tumor effect of group (4) CDDP+C200R108 is the best. The growth and proliferation of tumor cells are significantly inhibited, and the tumor volume is significantly reduced. It can be seen that increasing the dose of CpG1018 within a certain range has a better anti-tumor effect.

[0243] 13.3 Survival Curves of Tumor-Bearing Mice During Treatment

[0244] As shown in Figure 35, all mice in the saline control group died before the second round of dosing. After two dosing cycles, the survival rates of the 50μg, 100μg, and 200μg CpG1018 treatment groups were 60%, 100%, and 100%, respectively. By the end of the three dosing cycles, the survival rates were 20%, 60%, and 100%, respectively. Comparing the survival of the three groups of mice demonstrates that increasing the dose of CpG1018 significantly improves both the survival period and the survival rate of melanoma-bearing mice.

[0245] In this example, when the cisplatin dose was 0.4 mg / kg and the R848 dose remained unchanged, the CpG1018 dose was increased to 200 μg per mouse. From the perspective of the general condition and weight changes of the mice, no enhanced side effects were observed. This demonstrates that when other conditions remain unchanged, increasing the CpG1018 dose can enhance the tumor inhibition effect and prolong the survival of tumor-bearing mice.

[0246] Example 14 Therapeutic Effect of Cisplatin Combined with CR108 Tumor Adjuvant and Anti-PD-1 Antibody

[0247] This example further explored the effects of cisplatin combined with CR108 adjuvant and anti-PD-1 antibody on 4T-1 breast cancer model mice. 5 The number of tumor cells / mouse was subcutaneously inoculated to establish a mouse 4T-1 breast cancer model. The tumor volume was about 20 mm on the 5th day after inoculation. 3 ~30mm 3 Perform medication administration.

[0248] Specific mouse dosing groups: (1) PBS negative control group, (2) CDDP + CR108 (cisplatin 2 μg / kg, CpG1018 50 μg / mouse, water-soluble R848 50 μg / mouse), (3) CDDP + CR108 + αPD-1 mAb (cisplatin 2 μg / kg, CpG1018 50 μg / mouse, water-soluble R848 50 μg / mouse, anti-PD-1 antibody 200 μg / mouse), (4) αPD-1 mAb (anti-PD-1 antibody 200 μg / mouse). Except for the anti-PD-1 antibody, which was injected intraperitoneally, all other drugs were injected subcutaneously near the tumor. InVivoMAb anti-mouse PD-1 (CD279) was from BioXcell, product number BE0273.

[0249] Dosage time: The day of tumor implantation is counted as day 0.

[0250] (1) PBS group: 100 μl of PBS was administered on the 5th, 7th, 14th, and 21st days respectively;

[0251] (2) CDDP+CR108 group: 100 μl of CDDP was administered on day 5, and 100 μl of CR108 mixed drug was administered on days 7, 14, and 21;

[0252] (3) CDDP+CR108+αPD-1mAb group: 100 μl of CDDP was administered on day 5; 100 μl of CR108 mixed drug was administered on day 7, day 14, and day 21; 100 μl of anti-PD-1 antibody drug was administered on day 6, day 10, day 14, day 18, and day 22;

[0253] (4) αPD-1mAb group: 100ul of anti-PD-1 antibody drug was administered on the 6th, 10th, 14th, 18th, and 22nd days.

[0254] Conclusion: By measuring the tumor volume, as shown in Figure 36, the tumor growth of 4T-1 breast cancer model mice treated with Anti PD-1 antibody alone (αPD-1mAb group) was slowed down. In comparison, the CDDP+CR108 tumor adjuvant treatment group and the CDDP+CR108 tumor adjuvant combined with Anti PD-1 antibody treatment group (CDDP+CR108+αPD-1mAb group) significantly inhibited tumor growth, and there was no significant difference in the effects of the two groups on mouse tumor growth.

[0255] In summary, the low-dose cisplatin combined with CpG and R848 therapeutic composition provided by the present invention not only has an excellent inhibitory effect on the growth and proliferation of triple-negative breast cancer 4T1 cells, but also can inhibit the growth and proliferation of melanoma B16 cells, indicating that the combined therapeutic composition has a broad-spectrum inhibitory effect on tumor growth; moreover, the combined therapeutic composition induces the activation of B cells and T cells in tumor mice, promotes the levels of anti-tumor cytokines IFNγ, TNFα, IFNα, perforin and granzyme, and induces a large number of CD8 + T cells, CD4 + T cells, Tfh cells and B cells enter the tumor tissue to play an anti-tumor effect, which is significantly better than other groups, indicating that the combined therapeutic composition has an excellent effect in activating B cell and T cell immune responses, enabling more B cells and T cells to exercise effector functions, thereby inhibiting tumor growth; at the same time, the present invention provides a combined treatment method of the combined therapeutic composition, for small-volume tumors, that is, after using low-dose cisplatin, the mixed drug of CpG and R848 is started to be administered at an interval of about 2 days, and then the mixed drug of CpG and R848 is continuously administered multiple times at an interval of 7 days; for large-volume tumors, that is, a certain dose of cisplatin is first injected into the tumor in each administration cycle, and the mixed drug of CpG and R848 is injected into the tumor at an interval of about 2 days. The drug was taken once every other day for a cycle of four times, and the next dose was repeated after one week of discontinuation. This combined treatment method inhibited tumor growth most significantly compared to the other dosing method groups, indicating that the combined treatment method provided by the present invention has a better effect in inhibiting tumor growth. It was also found that the combined treatment composition and its combined treatment method provided by the present invention can not only significantly inhibit proximal tumor growth, but also significantly inhibit distal tumor growth, indicating that the combined treatment composition and its combined treatment method have an inhibitory effect on systemic tumor lesions, and further illustrates that the combined treatment composition effectively activates systemic anti-tumor immune responses and reactions, and is not only effective against local tumor lesions, but also has an efficient anti-tumor immune effect on tumor lesions that spread throughout the body. The above effects are applicable to scientific research-grade R848 (water-soluble) and medicinal R848 (lipid-soluble), scientific research-grade CpG1826 and medicinal CpG1018.

[0256] Therefore, the present invention provides a combined therapeutic composition and a combined treatment method for tumor treatment, wherein the combined therapeutic composition is a low-dose chemotherapy drug cisplatin combined with CpG and R848; the combined treatment method is to use cisplatin, CpG and R848 at a specific time, that is, first use a low-dose chemotherapy drug cisplatin to kill the tumor to release tumor neoantigens, allowing the body's immune system to independently recognize and screen the neoantigens, and then use CpG and R848 immune activators to induce and promote the body to produce tumor neoantigen-specific immune responses, enhance and strengthen the anti-tumor function of T cells, thereby achieving the effect of inhibiting tumor growth.

[0257] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a therapeutically effective amount of a platinum chemotherapy drug, a therapeutically effective amount of a CpG oligonucleotide and a therapeutically effective amount of R848.

2. The pharmaceutical composition according to claim 1, wherein the platinum chemotherapy drug is selected from one or more of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin; preferably, the platinum chemotherapy drug is cisplatin.

3. The pharmaceutical composition according to claim 1, wherein the therapeutically effective amount of CpG oligonucleotide (CpG ODN) is a class B CpG ODN or a class C CpG ODN; Preferably, the class B CpG ODN is selected from one or more of ODN 1826, ODN 1018, and ODN 2006 / 7909; Preferably, the C-class CpG ODN is selected from one or more of ODN M362, ODN 2395, and D-SL03.

4. The pharmaceutical composition according to claim 1, wherein the therapeutically effective amount of R848 is selected from one or more of water-soluble R848 and fat-soluble R848.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the therapeutically effective amount of the platinum chemotherapy drug is administered at a dosage of 2 μg / kg to 4000 μg / kg; The therapeutically effective amount of the CpG oligonucleotide is administered in a dosage of 0.001 mg / time to 32 mg / time; The therapeutically effective amount of R848 is administered in a dosage of 0.001 mg / time to 5 mg / time; Preferably, the therapeutically effective amount of the platinum chemotherapy drug is administered in a dosage of 2 μg / kg to 400 μg / kg; The therapeutically effective amount of the CpG oligonucleotide is administered in a dosage of 0.05 mg / time to 2 mg / time; The therapeutically effective amount of R848 is administered in a dosage of 0.01 mg / time to 1 mg / time.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the therapeutically effective amount of the platinum chemotherapy drug, the therapeutically effective amount of the CpG oligonucleotide, and the therapeutically effective amount of R848 are administered to a subject in need thereof in this order; Alternatively, the therapeutically effective amount of CpG oligonucleotide and the therapeutically effective amount of R848 are administered simultaneously, and the therapeutically effective amount of platinum chemotherapy drug is administered before the therapeutically effective amount of CpG oligonucleotide and the therapeutically effective amount of R848.

7. The pharmaceutical composition according to claim 6, wherein the therapeutically effective amount of the platinum chemotherapy drug is administered once, and the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are administered 1 to 5 times as one administration cycle; The time interval between the first administration of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 and the first administration of the therapeutically effective amount of the platinum chemotherapy drug is 1 to 5 days; Preferably, the first administration of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 is separated from the first administration of the therapeutically effective amount of the platinum chemotherapy drug by 2 days; wherein the therapeutically effective amount of CpG oligonucleotide and the therapeutically effective amount of R848 are administered for the first to fifth times at intervals of 1 to 14 days; The administration cycle for the subject is 1 to 3 times, with an interval of 5 to 7 days between each administration cycle.

8. The pharmaceutical composition according to any one of claims 1 to 7, characterized in that The administration of the pharmaceutical composition includes one or more of subcutaneous administration near the tumor and intratumoral injection.

9. The pharmaceutical composition according to claim 1, characterized in that It further comprises other tumor therapeutic drugs. Preferably, the other tumor therapeutic drugs are immunotherapeutic drugs. More preferably, the immunotherapeutic drugs are PD-1 / PD-L1 inhibitors.

10. A medicine kit for treating tumors or cancers, characterized in that: It includes a therapeutically effective amount of a platinum chemotherapy drug, a therapeutically effective amount of a CpG oligonucleotide, and a therapeutically effective amount of R848; Preferably, the platinum chemotherapy drug is selected from one or more of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin; preferably, the platinum is cisplatin; the therapeutically effective amount of CpG oligonucleotide (CpG ODN) is a class B CpG ODN or a class C CpG ODN; More preferably, the class B CpG ODN is selected from one or more of ODN 1826, ODN 1018, and ODN 2006 / 7909; More preferably, the C-class CpG ODN is selected from one or more of ODN M362, ODN 2395, and D-SL03; The therapeutically effective amount of R848 is selected from one or more of water-soluble R848 and fat-soluble R848.

11. The drug kit for treating tumors or cancers according to claim 10, characterized in that: The therapeutically effective amount of the platinum chemotherapy drug, the therapeutically effective amount of the CpG oligonucleotide, and the therapeutically effective amount of R848 are packaged separately, or the therapeutically effective amount of the platinum chemotherapy drug is packaged separately, and the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are mixed and packaged; Preferably, the therapeutically effective amount of the platinum chemotherapy drug, the therapeutically effective amount of the CpG oligonucleotide, and the therapeutically effective amount of R848 are sequentially administered to a subject in need thereof; More preferably, the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are administered simultaneously, and the therapeutically effective amount of the platinum chemotherapy drug is administered before the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848; More preferably, the therapeutically effective amount of the platinum chemotherapy drug is administered once, and the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are administered 1 to 5 times as one administration cycle; The time interval between the first administration of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 and the first administration of the therapeutically effective amount of the platinum chemotherapy drug is 1 to 5 days; Preferably, the first administration of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 is separated from the first administration of the therapeutically effective amount of the platinum chemotherapy drug by 2 days; wherein the therapeutically effective amount of CpG oligonucleotide and the therapeutically effective amount of R848 are administered for the first to fifth times at intervals of 1 to 14 days; The therapeutically effective amount of CpG oligonucleotide and the therapeutically effective amount of R848 are administered to the subject in a cycle of 1 to 3 times, with an interval of 5 to 7 days between each administration cycle.

12. The drug kit for treating tumors or cancers according to claim 10, characterized in that: It further comprises other tumor therapeutic drugs. Preferably, the other tumor therapeutic drugs are immunotherapeutic drugs. More preferably, the immunotherapeutic drugs are PD-1 / PD-L1 inhibitors.

13. Use of the pharmaceutical composition according to claims 1 to 9, or the medicine kit according to claims 10 to 12, in the preparation of a medicament for treating tumors or cancers.

14. The method according to claim 13, wherein The tumor or cancer is selected from breast cancer, melanoma, liver cancer, basal cell carcinoma, cutaneous squamous cell carcinoma, cutaneous T-cell lymphoma, and colorectal cancer; preferably, the tumor or cancer is selected from breast cancer and melanoma.

15. A method for treating tumors or cancers, characterized in that: The method comprises administering to a subject a therapeutically effective amount of a platinum chemotherapy drug, a therapeutically effective amount of a CpG oligonucleotide, and a therapeutically effective amount of R848; Preferably, the platinum chemotherapy drug is selected from one or more of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin; preferably, the platinum is cisplatin; the therapeutically effective amount of CpG oligonucleotide (CpG ODN) is a class B CpG ODN or a class C CpG ODN; More preferably, the class B CpG ODN is selected from one or more of ODN 1826, ODN 1018, and ODN 2006 / 7909; More preferably, the C-class CpG ODN is selected from one or more of ODN M362, ODN 2395, and D-SL03; The therapeutically effective amount of R848 is selected from one or more of water-soluble R848 and fat-soluble R848.

16. The method for treating tumors or cancers according to claim 15, wherein: The therapeutically effective amount of the platinum chemotherapy drug is administered in a dosage of 2 μg / kg to 4000 μg / kg; The therapeutically effective amount of the CpG oligonucleotide is administered in a dosage of 0.001 mg / time to 32 mg / time; The therapeutically effective amount of R848 is administered in a dosage of 0.001 mg / time to 5 mg / time; Preferably, the therapeutically effective amount of the platinum chemotherapy drug is administered in a dosage of 2 μg / kg to 400 μg / kg; The therapeutically effective amount of the CpG oligonucleotide is administered in a dosage of 0.05 mg / time to 2 mg / time; The therapeutically effective amount of R848 is administered in a dosage of 0.01 mg / time to 1 mg / time.

17. The method for treating tumors or cancers according to claim 16, wherein: The therapeutically effective amount of the platinum chemotherapy drug, the therapeutically effective amount of the CpG oligonucleotide, and the therapeutically effective amount of R848 are packaged separately, or the therapeutically effective amount of the platinum chemotherapy drug is packaged separately, and the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are mixed and packaged; Preferably, the therapeutically effective amount of the platinum chemotherapy drug, the therapeutically effective amount of the CpG oligonucleotide, and the therapeutically effective amount of R848 are sequentially administered to a subject in need thereof; More preferably, the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are administered simultaneously, and the therapeutically effective amount of the platinum chemotherapy drug is administered before the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848; More preferably, the therapeutically effective amount of the platinum chemotherapy drug is administered once, and the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 are administered 1 to 5 times as one administration cycle; The time interval between the first administration of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 and the first administration of the therapeutically effective amount of the platinum chemotherapy drug is 1 to 5 days; Preferably, the first administration of the therapeutically effective amount of the CpG oligonucleotide and the therapeutically effective amount of R848 is separated from the first administration of the therapeutically effective amount of the platinum chemotherapy drug by 2 days; wherein the therapeutically effective amount of CpG oligonucleotide and the therapeutically effective amount of R848 are administered for the first to fifth times at intervals of 1 to 14 days; The therapeutically effective amount of CpG oligonucleotide and the therapeutically effective amount of R848 are administered to the subject in a cycle of 1 to 3 times, with an interval of 5 to 7 days between each administration cycle.

18. The method for treating tumors or cancer according to claim 16, wherein: The tumor or cancer is selected from breast cancer, melanoma, liver cancer, basal cell carcinoma, cutaneous squamous cell carcinoma, cutaneous T-cell lymphoma, and colorectal cancer; preferably, the tumor or cancer is selected from breast cancer and melanoma.

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