Application of glutathione in enhancing tumor immunity

Abstract

The invention relates to the technical field of biology, in particular to application of glutathione in enhancing tumor immunity. The invention discloses a novel application of glutathione in the aspect of improving tumor immunity. The research finds that glutathione can recover function damage caused by accumulation of 3-hydroxykynurenine (3HK) of T cells due to accumulation of chemotherapeutic drugs, the effect of sensitizing the combined treatment of chemical and immunotherapy is enhanced, and glutathione may become a force-giving drug for sensitizing the immunotherapy in the era of tumors which are widely used in the combined treatment of chemical and immunotherapy at present.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of glutathione in enhancing tumor immunity. Background Art

[0002] For the treatment of cancer, the combination of chemotherapy and immunotherapy is an important treatment method at present. A large number of preclinical literatures show that the efficacy of conventional chemotherapy, including anthracyclines, oxaliplatin, and taxanes, is much better in immunocompetent mouse models than in immunodeficient mouse models. This evidence indicates that chemotherapeutic drugs also play a significant role in immunomodulation and profoundly affect the immune cells infiltrating in tumors.

[0003] In view of the fact that the phenomenon of changing chemotherapy regimens is very common in clinical practice, clinical trial studies have shown that the order of drugs in chemotherapy regimens has different effects on immune cells and affects the effect of immunotherapy. Therefore, it is crucial to explore the effect of changing chemotherapy regimens on CD8 + T cells and to discover new drugs that can improve the effect of immunotherapy after changing chemotherapeutic drugs. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide the application of glutathione in enhancing tumor immunity.

[0005] The present invention provides the application of glutathione in the preparation of a preparation and / or a drug combination for enhancing tumor immunity.

[0006] Furthermore, the present invention provides the application of glutathione and its preparation in the preparation of a drug combination for enhancing tumor immunity after changing chemotherapeutic drugs.

[0007] Furthermore,

[0008] the chemotherapeutic drug includes at least one of pemetrexed, cisplatin, and / or paclitaxel;

[0009] the tumor includes at least one or a combination of two or more of lung cancer, breast cancer, liver cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck tumors, central nervous system tumors, gynecological tumors, and / or soft tissue sarcomas and their metastases;

[0010] In the glutathione and its preparation, the dosage form of the preparation includes injection, oral preparation, and / or liposome encapsulation.

[0011] The present invention provides a drug combination for enhancing tumor immunity, the raw materials of which include glutathione and other drugs for enhancing tumor immunity;

[0012] The other drugs for enhancing tumor immunity include at least one of immune checkpoint inhibitors, cytokine drugs, thymosin drugs, Toll-like receptor (TLR) agonists, TGF-β inhibitors, CCR antagonists, levamisole, isoprinosine, biological polysaccharides, and / or tumor vaccines.

[0013] Furthermore,

[0014] The immune checkpoint inhibitors include PD-1 / PD-L1 inhibitors and / or CTLA-4 inhibitors;

[0015] The cytokine drugs include interleukin (IL) and / or interferon; the interleukin includes but is not limited to IL-2; the interferon includes but is not limited to IFN-α and IFN-β;

[0016] The thymosin drugs include thymosin, thymopentin, thymalfasin (thymosin α1):

[0017] The Toll-like receptor (TLR) agonists include Imiquimod;

[0018] The TGF-β inhibitors include Galunisertib, Vactosertib;

[0019] The CCR antagonists include the CCR5 antagonist Maraviroc, the CCR2 / CCR5 dual antagonist BMS-813160;

[0020] The tumor vaccines include Bacillus Calmette-Guérin (BCG), personalized tumor vaccines;

[0021] Even further,

[0022] The PD-1 / PD-L1 inhibitors include at least one of small molecules or polypeptides that block the binding of PD-1 and PD-L1, PD-1 monoclonal antibodies, and / or PD-L1 monoclonal antibodies;

[0023] The small molecules or polypeptides that block the binding of PD-1 and PD-L1 include CA-170 (small molecule PD-L1 inhibitor), INCB-086550 (small molecule PD-L1 inhibitor), biphenyl derivatives (preventing PD-1:PD-L1 interaction), and / or polypeptide molecules designed for the PD-1:PD-L1 receptor-ligand interface;

[0024] The PD-1 antibody inhibitors include: Nivolumab (Opdivo) (a humanized IgG4 monoclonal antibody against PD-1, which restores the anti-tumor function of T cells by blocking the binding of PD-1 to its ligands PD-L1 and PD-L2), Cemiplimab (Libtayo) (a humanized IgG4 monoclonal antibody mainly used for cutaneous squamous cell carcinoma), Toripalimab (a humanized IgG4 monoclonal antibody used for various solid tumors), and / or Tislelizumab (a humanized IgG4 monoclonal antibody with a unique Fc segment modification that reduces antibody-dependent cell phagocytosis (ADCP) and avoids T cell exhaustion);

[0025] The PD-L1 antibody inhibitors include: Atezolizumab (Tecentriq) (a humanized IgG4 monoclonal antibody that restores the anti-tumor function of T cells by blocking the binding of PD-L1 to PD-1), Avelumab (Bavencio) (a humanized IgG1 monoclonal antibody that blocks the binding of PD-L1 to PD-1), Durvalumab (Imfinzi) (a humanized IgG1 monoclonal antibody);

[0026] In the pharmaceutical combination of the present invention, the concentration of glutathione in in vitro tests is 0.5 - 5 mM; and the concentration in in vivo experiments is 80 mg / kg.

[0027] The pharmaceutical combination of the present invention further comprises pharmaceutically acceptable excipients;

[0028] The pharmaceutically acceptable excipients include at least one or a combination of two or more of solvents, solubilizers, cosolvents, emulsifiers, disintegrants, stabilizers, plasticizers, penetration enhancers, and / or sustained-release agents;

[0029] The solvents include water or buffers; the solubilizers include surfactants and / or cyclodextrins;

[0030] The cosolvents include organic acids and their sodium salts, amide compounds, inorganic salts, and / or cyclodextrins and their derivatives;

[0031] The emulsifiers include surfactants, polysaccharides, phospholipids, proteins, and / or polyoxyethylene fatty alcohol ethers;

[0032] The disintegrants include starches, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, microcrystalline cellulose;

[0033] The stabilizers include antioxidants, chelating agents, cyclodextrins and their derivatives, and / or surfactants;

[0034] The plasticizer includes polyols, organic esters and / or natural polymer derivatives;

[0035] The penetration enhancer includes alcohol compounds, terpene compounds and / or fatty acid esters;

[0036] The sustained-release agent includes hydrophilic gel, ethyl cellulose, polyethylene, polyvinyl chloride, ethylene-vinyl acetate copolymer and / or polymethacrylate.

[0037] The present invention discloses a new use of glutathione in enhancing tumor immunity after changing chemotherapy drugs. The research of the present invention finds that glutathione can restore the functional impairment of T cells caused by the accumulation of 3-hydroxykynurenine (3HK) due to the accumulation of chemotherapy drugs, and sensitize the effect of immune combination therapy. In the era of tumors widely using immune combination therapy at present, glutathione may become a powerful drug for sensitizing immunotherapy. Description of the Drawings

[0038] Figure 1 Shows the flow chart of collecting peripheral blood before and after changing chemotherapy drugs for lung adenocarcinoma patients and the expression of T cell function molecules after changing chemotherapy drugs; wherein A is the flow chart of collecting peripheral blood before and after changing chemotherapy drugs for lung adenocarcinoma patients; B is the expression of T cell function molecules after changing chemotherapy drugs;

[0039] Figure 2 Shows the effect of changing chemotherapy drugs on the function of tumor-infiltrating T cells; wherein, A shows the growth of tumor volume in each group after removing the drug; B shows the expression of Ki67, a proliferation index of T cells infiltrating in tumors in the Ctrl group, SR group and MR group, and C shows the expression of T cell function molecules infiltrating in tumors in the Ctrl group, SR group and MR group;

[0040] Figure 3 Shows the enrichment of 3HK in CD8 + T cells in the MR group. A shows the KEGG enrichment analysis of 176 metabolites screened after excluding the differences between the up-regulated metabolites in the poor region of the MR group and tumor cell metabolites; B shows the analysis of the region where 3HK accumulates in T cells caused by changing chemotherapy drugs; C shows the content of 3HK in the chemotherapy drug change group and the single drug group; D shows the change of the content of 3HK in CD8 + T cells in the chemotherapy drug change group and the single drug group of patients' peripheral blood;

[0041] Figure 4 Shows the effect of 3HK on T cell function and the effect of GSH on 3HK in T cells. Among them, A shows the change of T cell function treated with 3HK; B shows the transcriptome sequencing results of the function of T cells treated with 3HK; C shows the change of 3HK in T cells in the NC group, SR group, MR group and MR-GSH group;

[0042] Figure 5 It shows the tumor growth caused by glutathione-controlled dressing change. Among them, A shows the treatment flow chart; B shows the changes in tumor volume of the Ctrl group, SR group, MR group, and MR+GSH group over the treatment time.

[0043] Figure 6 It shows the restoration of T cell function inhibition caused by glutathione-controlled dressing change. Among them, Figures A, B, and C show the expression of functional molecules in the Ctrl group, SR group, MR group, and MR+GSH group; D shows the expression of proliferation indexes in the Ctrl group, SR group, MR group, and MR+GSH group.

[0044] Figure 7 It shows that glutathione can enhance the anti-tumor function of the combination of chemotherapy and immunotherapy. Among them, A shows the treatment flow chart of the combination of glutathione and anti-PD1 monoclonal antibody; B shows the changes in tumor volume of the Ctrl group, MR group, MR+PD1 group, MR+GSH group, PD1 group, and MR+GSH+PD1 group over the treatment time.

[0045] Figure 8 It shows that the combination of glutathione and the combination of chemotherapy and immunotherapy can enhance the anti-tumor function of T cells. Among them, Figures A, B, and C show the expression of functional molecules in the Ctrl group, MR group, MR+PD1 group, MR+GSH group, PD1 group, and MR+GSH+PD1 group; D shows the expression of proliferation indexes in the Ctrl group, MR group, MR+PD1 group, MR+GSH group, PD1 group, and MR+GSH+PD1 group. Detailed implementation mode

[0046] The present invention provides the application of glutathione in enhancing tumor immunity. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications in this article without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0047] The term "at least one" means one or more, and "a plurality" means two or more. "At least one" or its similar expressions refer to any combination of these items, including any combination of single items (one or more) or plural items (one or more). For example, "at least one of a, b, or c", or "at least one of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single items (one or more) or multiple items (one or more) respectively.

[0048] The terms "comprising", "having", or "including", including the use of their grammatical synonyms, should generally be understood as open and non - restrictive, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context. The use of any and all examples or exemplary language in this article, such as "for example" or "including", is merely intended to better illustrate the present invention and does not limit the scope of the present invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present invention.

[0049] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any value inherently and inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0050] In the present invention, the monoclonal antibody in the present invention is the abbreviation of monoclonal antibody.

[0051] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market. The present invention will be further described below in conjunction with the examples:

[0052] Example 1 Application of Glutathione in Enhancing Anti - tumor Immunity

[0053] I. Replacement of Chemotherapy Drugs Impairs T - cell Function

[0054] Collect the peripheral blood of lung adenocarcinoma patients before and after replacing chemotherapy drugs ( Figure 1 A in it), after removing red blood cells by erythrolysis, use flow - cytometry antibodies to detect CD8 + T - cell function molecules (GZMB, IL2, and TNFα). The flow - cytometry results show that after replacing chemotherapy drugs, the expression of T - cell function molecules decreases ( Figure 1 B in it).

[0055] To further verify this phenomenon, mice were divided into three groups: Ctrl, SR (single regimen) group, and MR (multiple regimens) group. LLC cells were subcutaneously inoculated into C57 mice to form tumors. After tumor formation, the SR group was given four intraperitoneal injections of pemetrexed + cisplatin (pemetrexed 50 mg / kg, cisplatin 2 mg / kg); after the MR group was given two doses of pemetrexed + cisplatin, it was changed to two doses of paclitaxel + cisplatin (paclitaxel 10 mg / kg, cisplatin 2 mg / kg). After the administration was completed, the expression of functional molecules and inhibitory molecules of CD8 + T cells infiltrating in the tumors was detected. The results showed that the expression of functional molecules (GZMB, IFNγ, TNFα) and the proliferation index Ki67 of T cells infiltrating in the tumors of the SR group increased ( Figure 2 B and C in). Although there was no obvious difference in the tumor curves between the SR group and the MR group after the administration was completed, after removing the drugs and extending the observation time, it was found that the growth of the tumor volume in the SR group was slower than that in the MR group ( Figure 2 A in). It is speculated that at the end of the administration, the effect of the chemotherapeutic drugs themselves on the tumor cells masked the differences in T cell functions. With the removal of the chemotherapeutic drugs, the differences in tumor volume caused by the differences in T cell functions were manifested. In summary, both the peripheral blood of patients and the mouse experiments showed that changing chemotherapeutic drugs could damage the functions of CD8 + T cells, up-regulate the expression of inhibitory molecules, and damage the immune function.

[0056] II. Changing chemotherapeutic drugs leads to the accumulation of 3HK in T cells

[0057] To further explore the mechanism by which changing chemotherapeutic drugs damages T cell functions, the LLC cell line of lung adenocarcinoma was transfused into mice via the tail vein to simulate a lung orthotopic tumor model. After tumor formation, the SR group was given four intraperitoneal injections of pemetrexed + cisplatin (pemetrexed 50 mg / kg, cisplatin 2 mg / kg); after the MR group was given two doses of pemetrexed + cisplatin, it was changed to two doses of paclitaxel + cisplatin (paclitaxel 10 mg / kg, cisplatin 2 mg / kg). After the administration was completed, the lung tumors and normal lung tissues were removed, frozen, and serially sectioned for spatial metabolomics sequencing, HE staining, and CD8 antibody immunohistochemical staining. According to the results of immunohistochemistry, the frozen sections were divided into cancer tissues (tumor tissue) and normal tissues (normal tissue); according to the results of CD8 antibody immunohistochemistry, the cancer tissue areas were further divided into CD8 + T cell enrichment regions (CD8 + T rich) and CD8 + T cell non-enrichment regions (CD8 +T poor), where CD8 + The T rich region contains more T cell metabolites, and CD8 + The T poor region contains more tumor cell metabolites. Excluding the metabolites upregulated in the poor region of the MR group, that is, excluding the differences in tumor cell metabolites, 176 metabolites were screened out for KEGG enrichment analysis, and it was found that tryptophan metabolism was highly enriched in the T cells of the MR group ( Figure 3 A in), where 3-hydroxykynurenine (3-HK) was specifically enriched in CD8 in the MR group + The T rich region, and 3HK is a metabolite downstream of kynurenine ( Figure 3 B in). Therefore, activated CD8 + T cells were divided into four groups: the NC group served as a negative control; the PEM group was treated with pemetrexed (PEM, 5 nM) combined with cisplatin (200 nM) for 48 h; the PEM→PTX group was treated with pemetrexed (PEM, 5 nM) combined with cisplatin (200 nM) for 24 h, and then treated with paclitaxel (PTX, 100 nM) combined with cisplatin (200 nM) for 24 h; the PTX group was treated with paclitaxel (PTX, 100 nM) combined with cisplatin (200 nM) for 48 h. The research results showed that compared with the single drug group, the content of 3HK increased in the PEM→PTX group that changed the chemotherapy drug ( Figure 3 C in). To further verify, peripheral blood of patients at baseline (base line, BL), the single drug group (SR), and the multi-drug group (MR) was collected. The results showed that the content of 3HK in the CD8 + T cells in the peripheral blood of patients in the MR group increased ( Figure 3 D in).

[0058] III. 3HK accumulation can damage T cell function, leading to T cell exhaustion, and glutathione can reduce the content of 3HK in T cells

[0059] To explore the effect of 3HK on T cell function, T cells activated in vitro were treated with 3HK (200 μM), and the functional indexes of T cells (IL2, IFNγ, TNFα) were detected. It was found that the function of T cells treated with 3HK decreased ( Figure 4 A in). The results of transcriptome sequencing of T cells treated with 3HK showed that the exhaustion genes (Tox, Lag3, Tigit, etc.) of T cells treated with 3HK were significantly upregulated ( Figure 4 B in). To explore whether glutathione (GSH) can reduce the content of 3HK in the MR group, 1 mM GSH was added while changing the chemotherapy drug and treated for 24 h. Then, the content of 3HK in T cells was detected by ELISA. It was found that adding GSH while changing the chemotherapy drug could significantly reduce 3HK in T cells ( Figure 4In C) above. The above results suggest that 3HK can impair T cell function, leading to T cell exhaustion, and GSH can alleviate the accumulation of 3HK caused by changing drugs.

[0060] IV. Glutathione restores T cell function inhibition caused by changing drugs

[0061] To further verify that glutathione restores the anti-tumor function of T cells, glutathione (80 mg / kg) was added during the change of chemotherapy drugs in the mouse experiment. It was found that the tumor control in the group with glutathione added during the change of chemotherapy drugs was the best ( Figure 5 In B) above. Flow cytometry was used to detect the function of T cells infiltrating in the tumor and the expression of proliferation molecules. Compared with the MR group, it was found that the expression of functional molecules increased ( Figure 6 In A, B, and C) above, and the proliferation index also increased ( Figure 6 In D) above.

[0062] V. Glutathione can enhance the anti-tumor function of the combination of chemotherapy and immunotherapy

[0063] Since changing chemotherapy drugs can impair the anti-tumor function of T cells, we wanted to explore whether glutathione can sensitize the immunotherapy effect of PD1 monoclonal antibody. Therefore, glutathione and PD1 monoclonal antibody were added during the change of drugs, Figure 7 B in above shows that the tumor control of the combination of glutathione and chemotherapy-immunotherapy is the best. Flow cytometry was used to detect the function of T cells infiltrating in the tumor and the expression of proliferation. Compared with the MR+PD1 group, it was found that the expression of functional molecules in the MR+GSH+PD1 group increased ( Figure 8 In A, B, and C) above, and the proliferation index also increased ( Figure 8 In D) above. In summary, it shows that glutathione can restore the impairment of T cell function caused by changing chemotherapy drugs and enhance the effect of the combination of chemotherapy and immunotherapy.

[0064] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of glutathione and its preparations in the preparation of a drug combination for enhancing tumor immunity after changing chemotherapy drugs.

2. The application according to claim 1, wherein The chemotherapy drugs include at least one of pemetrexed, cisplatin, and / or paclitaxel.

3. The application according to claim 1, characterized in that, The tumors include at least one or a combination of two or more of lung cancer, breast cancer, liver cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck tumors, central nervous system tumors, gynecological tumors, and / or soft tissue sarcomas and their metastases.

4. The application according to claim 1, characterized in that, In the glutathione and its preparations, the dosage form of the preparations includes injection, oral preparation, and / or liposome encapsulation.

5. A pharmaceutical combination, characterized in that, The raw materials include glutathione and other drugs for enhancing tumor immunity; The other drugs for enhancing tumor immunity include at least one of immune checkpoint inhibitors, cytokine drugs, thymosin drugs, Toll-like receptor (TLR) agonists, TGF-β inhibitors, CCR antagonists, levamisole, isoprinosine, biological polysaccharides, and / or tumor vaccines.

6. The pharmaceutical combination according to claim 5, wherein, The concentration of the glutathione is 0.5 - 5 mM or 80 mg / kg.

7. The pharmaceutical combination according to claim 5, characterized in that, The immune checkpoint inhibitors include PD-1 / PD-L1 inhibitors and / or CTLA-4 inhibitors.

8. The pharmaceutical combination according to claim 7, characterized in that, The PD-1 / PD-L1 inhibitors include at least one of small molecules or polypeptides that block the binding of PD-1 and PD-L1, anti-PD-1 monoclonal antibodies, and / or anti-PD-L1 monoclonal antibodies.

9. The pharmaceutical combination according to claim 6 or 8, characterized in that, It also includes pharmaceutically acceptable excipients.

10. The pharmaceutical combination according to claim 9, characterized in that, The pharmaceutically acceptable excipients include at least one or a combination of two or more of solvents, solubilizers, cosolvents, emulsifiers, disintegrants, stabilizers, plasticizers, penetration enhancers, and / or sustained-release agents.

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