A composition for antitumor purposes and its application

Abstract

This invention discloses an anti-tumor composition and its application, relating to the field of biomedical technology. The composition comprises an inhibitor of anti-immune checkpoints and an inhibitor of the expression and / or activity of the RBM10 gene. Research in this invention has found that reducing the expression level of the RBM10 gene in lung cancer cells using substances such as shRNA can significantly enhance the therapeutic effect of the anti-immune checkpoint inhibitor on a mouse model of lung cancer, effectively inhibiting tumor growth; further combination with Eubacterium hallii and / or Akkermansia muciniphila can also effectively enhance the inhibitory effect on tumor growth.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a composition for anti-tumor purposes and its application. Background Technology

[0002] In recent years, the incidence of cancer has continued to rise, becoming a significant public health issue worldwide. Currently, the main treatments for cancer include targeted therapy, surgery, chemotherapy, and radiation therapy. Targeted therapy is expensive and has limited applicability, resulting in its relatively low clinical application. Chemotherapy is effective to some extent, but it often comes with significant side effects and drug resistance (e.g., causing nausea and vomiting, and in severe cases, infection and bleeding). Radiation therapy can also cause nausea and vomiting, and in severe cases, hair loss. Therefore, new treatment strategies are needed. Summary of the Invention

[0003] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a composition that can effectively treat cancers such as lung cancer.

[0004] The present invention also provides applications of the above composition.

[0005] The present invention also provides a medicine comprising the above-described composition.

[0006] The present invention also provides the use of at least one of the following: an inhibitor of RBM10 gene expression and / or activity, Eubacterium hallii, and Akkermansia muciniphila, in the preparation of an synergist for the preparation of an inhibitor of anti-immune checkpoints in tumor therapy.

[0007] The present invention also provides the use of at least one of an anti-immune checkpoint inhibitor, Eubacterium hallii, and Akkermansiamuciniphila in the preparation of a medicament for treating tumors containing inactive mutations of the RBM10 gene.

[0008] According to a first aspect of the present invention, a composition comprises an inhibitor of immune checkpoints and an inhibitor of the expression and / or activity of the RBM10 gene.

[0009] The composition according to embodiments of the present invention has at least the following beneficial effects:

[0010] Combining inhibitors of immune checkpoints with inhibitors of RBM10 gene expression and / or activity can significantly improve the growth inhibition effect on tumors such as lung cancer, which is superior to the effect of using either one alone.

[0011] According to some embodiments of the present invention, the immune checkpoints include at least one of PD-1, PD-L1, CTLA-4, PD-L2, IDO-1, TIM3, LAG-3, KIR, 4-1BB, OX40, B7-H3, B7-H4, CD160, CD39, CD73, A2aR, VISTA, arginase I, TIGIT, and CD115.

[0012] According to some embodiments of the present invention, the inhibitor of the anti-immune checkpoint includes at least one of antibodies and small molecule compounds.

[0013] According to some embodiments of the present invention, the inhibitor of the anti-immune checkpoint is a PD-1 antibody.

[0014] According to some embodiments of the present invention, the expression and / or activity inhibitors of the RBM10 gene include at least one of siRNA, antisense RNA, miRNA, and shRNA.

[0015] According to some embodiments of the present invention, the nucleotide sequence of the inhibitor of expression and / or activity of the RBM10 gene is GCAGTCCCATGGTGTCCAAGC.

[0016] According to some embodiments of the present invention, the expression and / or activity inhibitor of the RBM10 gene is shRNA; the nucleotide sequence of the sense strand of the shRNA is shown in SEQ ID NO:1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:2.

[0017] According to some embodiments of the present invention, the composition further includes at least one of Eubacterium hallii and Akkermansia muciniphila.

[0018] The use of the composition described in the first aspect of the second aspect of the present invention in the preparation of an antitumor drug.

[0019] According to some embodiments of the present invention, the tumor includes at least one of lung cancer, colon cancer, and breast cancer.

[0020] According to some embodiments of the present invention, the lung cancer is non-small cell lung cancer.

[0021] An antitumor drug according to a third aspect of the present invention comprises the composition described in the first aspect.

[0022] According to some embodiments of the present invention, the drug further includes pharmaceutically acceptable excipients.

[0023] The use of at least one of the following, an inhibitor of RBM10 gene expression and / or activity, Eubacterium hallii, and Akkermansia muciniphila, in the preparation of an synergist for the preparation of an inhibitor of anti-immune checkpoints for tumor treatment, according to a fourth aspect of the present invention.

[0024] The use of at least one of the following, an anti-immune checkpoint inhibitor, Eubacterium hallii, and Akkermansia muciniphila, in the preparation of a medicament for treating tumors containing inactive mutations of the RBM10 gene, according to a fifth aspect embodiment of the present invention.

[0025] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0026] Figure 1 This is a flowchart of the experiment in Example 2;

[0027] Figure 2 The therapeutic effects of sh-RBM10 and PD-1 inhibitors on a mouse subcutaneous tumor model in Example 2 are shown in Figure 2. A: Tumor photographs at the experimental endpoint; B: Tumor weight and volume at the experimental endpoint; C: Tumor growth curve (**** indicates a highly significant difference compared to the LLC-NC group, P<0.0001); D: Synergistic index calculation results.

[0028] Figure 3 This is a flowchart of the experiment in Example 3;

[0029] Figure 4 The therapeutic effects of sh-RBM10, PD-1 inhibitor, and E. hallii on a mouse subcutaneous tumor model in Example 3 are shown below. A: Tumor photographs at the experimental endpoint; B: Tumor weight and volume at the experimental endpoint (* indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001); C: Tumor growth curve (**** indicates a highly significant difference from the LLC-NC group, P<0.0001); D: Synergistic index calculation results.

[0030] Figure 5 This is a flowchart of the experiment in Example 4;

[0031] Figure 6This section describes the therapeutic effects of sh-RBM10, PD-1 inhibitors, and AKK on a mouse subcutaneous tumor model, as described in Example 4. A: Tumor photographs at the experimental endpoint; B: Tumor weight and volume at the experimental endpoint (* indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001); C: Tumor growth curve (**** indicates a highly significant difference compared to the LLC-NC group, P<0.0001); D: Synergy index calculation results.

[0032] Figure 7 This is a flowchart of the experiment in Example 5;

[0033] Figure 8 The therapeutic effects of sh-RBM10, PD-1 inhibitor, E. hallii, and AKK on a mouse subcutaneous tumor model in Example 5 are shown below. A: Tumor photographs at the experimental endpoint; B: Tumor weight and volume at the experimental endpoint (* indicates P<0.05, *** indicates P<0.001, **** indicates P<0.0001); C: Tumor growth curve (**** indicates a highly significant difference from the LLC-NC group, P<0.0001); D: Synergistic index calculation results. Detailed Implementation

[0034] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0035] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0036] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0037] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0038] According to a first aspect of the present invention, a composition comprises an inhibitor of immune checkpoints and an inhibitor of the expression and / or activity of the RBM10 gene.

[0039] According to some embodiments of the present invention, the immune checkpoints include at least one of PD-1, PD-L1, CTLA-4, PD-L2, IDO-1, TIM3, LAG-3, KIR, 4-1BB, OX40, B7-H3, B7-H4, CD160, CD39, CD73, A2aR, VISTA, arginase I, TIGIT, and CD115.

[0040] According to some embodiments of the present invention, the inhibitor of the anti-immune checkpoint includes at least one of antibodies and small molecule compounds.

[0041] According to some embodiments of the present invention, the inhibitor of the anti-immune checkpoint is a PD-1 antibody.

[0042] According to some embodiments of the present invention, the PD-1 antibody includes at least one of nivolumab, pembrolizumab, cimiprimab, toripalimab, sintilimab, and camrelizumab.

[0043] According to some embodiments of the present invention, the expression and / or activity inhibitors of the RBM10 gene include at least one of siRNA, antisense RNA, miRNA, and shRNA.

[0044] According to some embodiments of the present invention, the nucleotide sequence of the inhibitor of expression and / or activity of the RBM10 gene is GCAGTCCCATGGTGTCCAAGC.

[0045] According to some embodiments of the present invention, the expression and / or activity inhibitor of the RBM10 gene is shRNA; the shRNA includes a stem-loop structure connecting the sense and antisense strands of the siRNA, a sequence complementary to the sticky end of the vector restriction enzyme site, and a terminator.

[0046] According to some embodiments of the present invention, the expression and / or activity inhibitor of the RBM10 gene is shRNA; the nucleotide sequence of the sense strand of the shRNA is shown in SEQ ID NO:1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:2.

[0047] According to some embodiments of the present invention, the composition further includes at least one of Eubacterium hallii and Akkermansia muciniphila. Inhibitors of immune checkpoints, inhibitors of RBM10 gene expression and / or activity, combined with Eubacterium hallii and / or Akkermansia muciniphila, exhibit better antitumor effects.

[0048] According to some embodiments of the present invention, the composition further includes *Eubacterium hallii* and *Akkermansia muciniphila*; the ratio of viable bacteria counts of *Eubacterium hallii* and *Akkermansia muciniphila* is 1:(0.8 to 1.2). For example, it can be 1:0.8, 1:0.85, 1:0.9, 1:0.95, 1:1, 1:1.05, 1:1.1, 1:1.15, or 1:1.2.

[0049] The use of the composition described in the first aspect of the second aspect of the present invention in the preparation of an antitumor drug.

[0050] According to some embodiments of the present invention, the tumor includes at least one of lung cancer, colon cancer, and breast cancer.

[0051] According to some embodiments of the present invention, the lung cancer is non-small cell lung cancer.

[0052] An antitumor drug according to a third aspect of the present invention comprises the composition described in the first aspect.

[0053] According to some embodiments of the present invention, the drug further includes pharmaceutically acceptable excipients.

[0054] According to some embodiments of the present invention, the excipients include diluents (including but not limited to starches, sugars, celluloses, or inorganic salts), wetting agents (including but not limited to water), binders (including but not limited to starch paste, dextrin, sugars, cellulose derivatives, gelatin, povidone, or polyethylene glycol), disintegrants (including but not limited to starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, croscarmellose, or surfactants), lubricants (including but not limited to talc, calcium stearate, magnesium stearate, magnesium dodecyl sulfate, micronized silica gel, or polyethylene glycol), color and flavor modifiers (including but not limited to pigments, fragrances, sweeteners, adhesives, or deodorants), solvents (including but not limited to water, ethanol, glycerin, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oils, or ethyl acetate), solubilizers (including but not limited to Tween compounds, malt extracts, polyoxyethylene fatty alcohol ethers, soaps, sulfates, or sulfonates), and cosolvents (including but not limited to organic acids and their salts). Amides and amines, inorganic salts, polyethylene glycol, povidone or glycerin), emulsifiers (including but not limited to: Span, Tween, maltose, benzyl esters, glycerol fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, tragacanth gum, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxides, silica, bentonite), antioxidants (including but not limited to: sulfites, metabisulfites, bisulfites, ascorbic acid, gallic acid or esters), metal complexing agents (including but not limited to: amides and amines, inorganic salts, polyethylene glycol, povidone or glycerin), emulsifiers (including but not limited to: Span, Tween, maltose, benzyl ester, glycerol fatty acid esters, higher fatty acid salts, sulfates, sulfides, gum arabic, astragalic acid or gallic acid or esters), metal complexing agents (including but not limited to: amides and amines, inorganic salts, polyethylene glycol, povidone or glycerin), emulsifiers (including but not limited to: Span, Tween, maltose, benzyl ester, glycerol fatty acid esters ... Limited to at least one of the following: disodium ethylenediaminetetraacetate or polycarboxylic acid compounds; preservatives (including but not limited to: parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols or phenols); pH adjusters (including but not limited to hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphates, acetates or citrates); and isotonic or isotropic adjusters (including but not limited to glucose, sodium chloride, sodium citrate, sorbitol or xylitol).

[0055] According to some embodiments of the present invention, the dosage form of the drug includes pills, tablets, granules, capsules, powders, suspensions, oral liquids, tube feeding preparations, or enemas. It is understood that the drug can be prepared into a suitable dosage form according to clinical needs.

[0056] According to some embodiments of the present invention, the drug is administered via oral administration, enema administration, or parenteral administration. It is understood that the drug can be administered via a suitable route of administration according to clinical needs.

[0057] According to some embodiments of the present invention, the drug administration cycle includes intermittent administration, periodic administration, continuous administration, or long-term administration. It is understood that the drug can be administered using a suitable administration cycle based on clinical needs.

[0058] Administration of the compositions herein includes, but is not limited to, administration at a therapeutically effective amount of the compositions described herein. The term "therapeutically effective amount" refers to the amount of a therapeutic agent that can be used to treat and / or prevent conditions that can be treated by administration of the compositions described herein. This dose is sufficient to demonstrate a therapeutic, preventative, or ameliorative effect. Such effects include the treatment and / or prevention of the conditions listed herein. The precise effective amount for a subject will depend on the subject's weight and health status, the nature and severity of the condition to be treated, the advice of the treating physician, and the chosen method or combination of treatments for administration.

[0059] According to some embodiments of the present invention, the drug is administered to humans or mice, among other things.

[0060] The use of at least one of the following, an inhibitor of RBM10 gene expression and / or activity, Eubacterium hallii, and Akkermansia muciniphila, in the preparation of an synergist for the preparation of an inhibitor of anti-immune checkpoints for tumor treatment, according to a fourth aspect of the present invention.

[0061] The use of at least one of the following, an anti-immune checkpoint inhibitor, Eubacterium hallii, and Akkermansia muciniphila, in the preparation of a medicament for treating tumors containing inactive mutations of the RBM10 gene, according to a fifth aspect embodiment of the present invention.

[0062] In the embodiments described herein, the PD-1 inhibitor used was purchased from BioXCell, catalog number BE0146; Eubacterium hallii (E. hallii) was purchased from DSMZ, catalog number DSM 3353; and Akkermansia muciniphila (AKK) was purchased from ATCC, catalog number ATCC BAA-835.

[0063] In the embodiments described herein, unless otherwise specified, the relative tumor volume is calculated by using the ratio of the experimental endpoint tumor volume of the treatment group to that of the control group. A synergy index less than 0 indicates a synergistic effect, a synergistic index equal to 0 indicates an additive effect, and a synergistic index greater than 0 indicates an antagonistic effect. For the specific calculation process, please refer to the literature "Assessing Interactions for Fixed-Dose Drug Combinations in Tumor Xenograft Studies" (Jianrong, Wu, Lorraine, et al. Assessing Interactions for Fixed-Dose Drug Combinations in Tumor Xenograft Studies[J]. Journal of BiopharmaceuticalStatistics, 2012. DOI:10.1080 / 10543406.2011.556285.).

[0064] Example 1

[0065] 1. Design and synthesis of shRNA:

[0066] shRNAs were designed targeting a specific site in the mouse RBM10 gene (Genbank: NM_145627). The nucleotide sequence of the sense strand of the shRNA is shown in SEQ ID NO:1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:2. The nucleotide sequence of the sense strand of the control shRNA is shown in SEQ ID NO:3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:4.

[0067] sh-RBM10-F: ccggGCAGTCCCATGGTGTCCAAGCctcgagGCTTGGACACCATGGGACTGCtttttg (SEQ ID NO: 1);

[0068] Wherein, ccgg is the AgeI restriction site added at the 5' end, ctcgag is the loop sequence, ttttt is the terminator sequence added at the 3' end, and g is the remaining EcoRI restriction site at the 3' end.

[0069] sh-RBM10-R:aattcaaaaaGCAGTCCCATGGTGTCCAAGCctcgagGCTTGGACACCATGGGACTGC (SEQ ID NO: 2).

[0070] NC-F: ccggTTCTCCGAACGTGTCACGTctcgagACGTGACACGTTCGGAGAAtttttg (SEQ ID NO: 3).

[0071] NC-R: aattcaaaaaTTCTCCGAACGTGTCACGTctcgagACGTGACACGTTCGGAGAA (SEQ ID NO: 4).

[0072] The sense and antisense strands were synthesized chemically. Double-stranded shRNAs (sh-RBM10 and sh-NC) were obtained through annealing (the synthesized paired DNA powder was dissolved in annealing buffer, incubated at 95°C for 15 min, and then allowed to cool to room temperature). The GV493 vector was digested with restriction endonucleases AgeI and EcoRI to obtain a linear vector. The linear vector and double-stranded shRNA were ligated using T4 DNA ligase to obtain the ligation product. The ligation product was transformed into *E. coli* DH5α competent cells. Positive clones were screened by PCR and sequencing. After amplification, lentiviral plasmids containing sh-RBM10 and sh-NC were extracted.

[0073] 2. Packaging of lentiviruses:

[0074] HEK293T cells in logarithmic growth phase were divided into groups of 5 × 10⁻⁶. 6 Cells were seeded per well into 10cm cell culture dishes and cultured until cell conjugation reached 80%. Transfection reagent was then used. (Supplier: Polyplus, Catalog No. 01000046) Lentiviral plasmids were transfected into HEK293T cells. Cell culture supernatants were collected, and lentiviruses containing sh-RBM10 (viral titer: 3 × 10⁻⁶) were collected by ultracentrifugation. 9 PFU / mL) and lentiviruses containing sh-NC (viral titer of 5 × 10⁻⁶). 8 PFU / mL).

[0075] 3. Evaluation of shRNA interference efficiency:

[0076] LLC cells were administered at a rate of 1×10 4Cells were seeded into 96-well plates and cultured for 24 h. Then, they were infected with lentivirus containing sh-RBM10 at an MOI of 300. After 72 h of culture, LLC cells with silenced RBM10 genes (hereinafter referred to as "LLC-sh-RBM10") were obtained. Control LLC cells (hereinafter referred to as "LLC-NC") were obtained using the same treatment method with lentivirus containing sh-NC. The expression level of RBM10 gene was detected by qRT-PCR using β-Actin gene as an internal control to evaluate the interference efficiency of sh-RBM10.

[0077] sh-RBM10 can efficiently silence the RBM10 gene, with a silencing efficiency of 85.56%.

[0078] Example 2

[0079] Twenty C57BL / 6 mice (6–8 weeks old) were randomly divided into four groups to establish a mouse subcutaneous tumor model. Mice in each group received different treatments, as detailed in the protocol below. Figure 1 .

[0080] (1) Control group (LLC-NC): 5×10⁻⁶ cells were injected subcutaneously. 6 LLC-NC cells obtained in Example 1;

[0081] (2) LLC-sh-RBM10 group: 5×10⁻⁶ injections were administered subcutaneously. 6 LLC-sh-RBM10 cells obtained in Example 1;

[0082] (3) aPD-1+LLC-NC group: subcutaneous injection of 5×10 6 The LLC-NC cells obtained in Example 1 were injected intraperitoneally with 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) on the fifth day;

[0083] (4) aPD-1+LLC-sh-RBM10 group: subcutaneous injection of 5×10 6 The LLC-sh-RBM10 cells obtained in Example 1 were injected intraperitoneally with 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) on the fifth day.

[0084] Tumor volume was measured every 2 days. On day 20, the mice were euthanized, the tumors were removed, and the tumor weight and volume at the experimental endpoint were measured to calculate the synergistic index.

[0085] Synergy index = log(relative tumor volume of aPD-1+LLC-sh-RBM10 group) - log(relative tumor volume of LLC-sh-RBM10 group) - log(relative tumor volume of aPD-1+LLC-NC group).

[0086] The results are as follows Figure 2 As shown.

[0087] The combination of PD-1 inhibitors and sh-RBM10 has a synergistic anti-tumor effect (synergistic index of -0.25), which is significantly superior to the monotherapy group.

[0088] Example 3

[0089] Thirty C57BL / 6 mice (6–8 weeks old) were randomly divided into six groups to establish a mouse subcutaneous tumor model. Mice in each group received different treatments, as detailed in the protocol below. Figure 3 .

[0090] (1) Control group (LLC-NC): 5×10⁻⁶ cells were injected subcutaneously. 6 LLC-NC cells obtained in Example 1;

[0091] (2) LLC-sh-RBM10 group: 5×10⁻⁶ injections were administered subcutaneously. 6 LLC-sh-RBM10 cells obtained in Example 1;

[0092] (3) aPD-1+LLC-NC group: subcutaneous injection of 5×10 6 The LLC-NC cells obtained in Example 1 were injected intraperitoneally with 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) on the fifth day;

[0093] (4) aPD-1+LLC-sh-RBM10 group: subcutaneous injection of 5×10 6 The LLC-sh-RBM10 cells obtained in Example 1 were injected intraperitoneally with 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) on the fifth day.

[0094] (5) E. hallii + aPD-1 + LLC-NC group: subcutaneous injection of 5×10 6 LLC-NC cells obtained in Example 1 were administered via gavage with 0.1 mL of E. halliformis suspension (2 × 10⁻⁶). 8 On day 5, 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) was injected intraperitoneally.

[0095] (6) E. hallii + aPD-1 + LLC-sh-RBM10 group: subcutaneous injection of 5×10 6 LLC-sh-RBM10 cells obtained in Example 1 were administered via gavage with 0.1 mL of E. hallii bacterial suspension (2 × 10⁻⁶). 8 On day 5, 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) was injected intraperitoneally.

[0096] Tumor volume was measured every 2 days. On day 20, the mice were euthanized, the tumors were removed, and the tumor weight and volume at the experimental endpoint were measured to calculate the synergistic index.

[0097] Synergy index = log(relative tumor volume of E.hallii+aPD-1+LLC-sh-RBM10 group) - log(relative tumor volume of LLC-sh-RBM10 group) - log(relative tumor volume of E.hallii+aPD-1+LLC-NC group).

[0098] The results are as follows Figure 4 As shown.

[0099] The combination of PD-1 inhibitors, sh-RBM10, and E. hallii has a synergistic anti-tumor effect (synergistic index of -1.25), which has a significant advantage over the single-drug group or the two-drug combination group.

[0100] Example 4

[0101] Thirty C57BL / 6 mice (6–8 weeks old) were randomly divided into six groups to establish a mouse subcutaneous tumor model. Mice in each group received different treatments, as detailed in the protocol below. Figure 5 .

[0102] (1) Control group (LLC-NC): 5×10⁻⁶ cells were injected subcutaneously. 6 LLC-NC cells obtained in Example 1;

[0103] (2) LLC-sh-RBM10 group: 5×10⁻⁶ injections were administered subcutaneously. 6 LLC-sh-RBM10 cells obtained in Example 1;

[0104] (3) aPD-1+LLC-NC group: subcutaneous injection of 5×10 6 The LLC-NC cells obtained in Example 1 were injected intraperitoneally with 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) on the fifth day;

[0105] (4) aPD-1+LLC-sh-RBM10 group: subcutaneous injection of 5×106 The LLC-sh-RBM10 cells obtained in Example 1 were injected intraperitoneally with 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) on the fifth day.

[0106] (5) AKK+aPD-1+LLC-NC group: subcutaneous injection of 5×10 6 LLC-NC cells obtained in Example 1 were administered by gavage with 0.1 mL of AKK bacterial suspension (2 × 10⁻⁶). 8 On day 5, 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) was injected intraperitoneally.

[0107] (6) AKK+aPD-1+LLC-sh-RBM10 group: 5×10⁻⁶ injections subcutaneously. 6 LLC-sh-RBM10 cells obtained in Example 1 were administered 0.1 mL of AKK bacterial suspension (2 × 10⁻⁶) by gavage. 8 On day 5, 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) was injected intraperitoneally.

[0108] Tumor volume was measured every 2 days. On day 20, the mice were euthanized, the tumors were removed, and the tumor weight and volume at the experimental endpoint were measured to calculate the synergistic index.

[0109] Synergy index = log(relative tumor volume of AKK+aPD-1+LLC-sh-RBM10 group) - log(relative tumor volume of LLC-sh-RBM10 group) - log(relative tumor volume of AKK+aPD-1+LLC-NC group).

[0110] The results are as follows Figure 6 As shown.

[0111] The combination of PD-1 inhibitors, sh-RBM10, and AKK has a synergistic anti-tumor effect (synergistic index of -0.09), which has a significant advantage over the single-drug group or the two-drug combination group.

[0112] Example 5

[0113] Thirty C57BL / 6 mice (6–8 weeks old) were randomly divided into six groups to establish a mouse subcutaneous tumor model. Mice in each group received different treatments, as detailed in the protocol below. Figure 7 .

[0114] (1) Control group (LLC-NC): 5×10⁻⁶ cells were injected subcutaneously. 6 LLC-NC cells obtained in Example 1;

[0115] (2) LLC-sh-RBM10 group: 5×10⁻⁶ injections were administered subcutaneously. 6 LLC-sh-RBM10 cells obtained in Example 1;

[0116] (3) aPD-1+LLC-NC group: subcutaneous injection of 5×10 6 The LLC-NC cells obtained in Example 1 were injected intraperitoneally with 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) on the fifth day;

[0117] (4) aPD-1+LLC-sh-RBM10 group: subcutaneous injection of 5×10 6 The LLC-sh-RBM10 cells obtained in Example 1 were injected intraperitoneally with 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) on the fifth day.

[0118] (5) AKK+E. hallii+aPD-1+LLC-NC group: subcutaneous injection of 5×10 6 LLC-NC cells obtained in Example 1 were administered via gavage with 0.2 mL of bacterial suspension (AKK and E. hallii, 2 × 10⁻⁶ each). 8 On day 5, 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) was injected intraperitoneally.

[0119] (6) AKK+E.hallii+aPD-1+LLC-sh-RBM10 group: subcutaneous injection of 5×10 6 LLC-sh-RBM10 cells obtained in Example 1 were administered via gavage with 0.2 mL of bacterial suspension (AKK and E. hallii, 2 × 10⁻⁶ each). 8 On day 5, 0.2 mL of PD-1 inhibitor (0.75 mg / mL, every 3 days) was injected intraperitoneally.

[0120] Tumor volume was measured every 2 days. On day 20, the mice were euthanized, the tumors were removed, and the tumor weight and volume at the experimental endpoint were measured to calculate the synergistic index.

[0121] Synergy index = log(relative tumor volume of AKK+E.hallii+aPD-1+LLC-sh-RBM10 group) - log(relative tumor volume of LLC-sh-RBM10 group) - log(relative tumor volume of AKK+E.hallii+aPD-1+LLC-NC group).

[0122] The results are as follows Figure 8 As shown.

[0123] The combination of PD-1 inhibitors, sh-RBM10, E. hallii, and AKK showed synergistic anti-tumor effects (synergistic index of -0.47), with significant advantages over the monotherapy group, the two-drug combination group, or the three-drug combination group.

[0124] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A composition, characterized in that, The composition comprises an inhibitor of immune checkpoints, an inhibitor of RBM10 gene expression and / or activity, Eubacterium hallii , Akkermansia muciniphila Composition; the immune checkpoint is PD 1; The expression and / or activity inhibitor of the RBM10 gene is shRNA; the nucleotide sequence of the sense strand of the shRNA is shown in SEQ ID NO: 1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO: 2; the inhibitor of the anti-immune checkpoint is an antibody.

2. The use of the composition of claim 1 in the preparation of an antitumor drug; wherein the tumor is non-small cell lung cancer.

3. An antitumor drug, characterized in that, The tumor includes the composition of claim 1; the tumor is non-small cell lung cancer.

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