Use of dimethyl fumarate to promote immunotherapy of renal cell carcinoma

Abstract

The present application belongs to the field of tumor treatment, and particularly relates to a pharmaceutical composition or kit, comprising a therapeutically effective amount of dimethyl fumarate (DMF) and a tumor immunotherapeutic agent and a pharmaceutically acceptable carrier. + The dimethyl fumarate can improve the protein level of PD-L1, further enhance the tumor therapeutic agent and CD8 The anti-tumor immune activity of T cells is improved, the accuracy of PD-L1 antibody targeting tumor cells is improved, the growth of tumor cells is significantly inhibited, the anti-tumor effect is improved, and it is suggested that DMF has potential clinical application value as an "sensitizer" for ICB treatment.

Description

Technical Field

[0001] This invention belongs to the field of tumor treatment, specifically relating to the use of dimethylfumaric acid (DMF) to promote immunotherapy for renal cell carcinoma. Background Technology

[0002] Renal cell carcinoma (RCC) is the most common malignant tumor of the urinary system, originating from the proximal tubular epithelium of the renal parenchyma, accounting for approximately 90% of all kidney tumors. Its pathogenesis is complex and often accompanied by specific genetic alterations, most notably VHL gene mutations or inactivation. RCC is typically highly aggressive and has a high risk of metastasis, especially in advanced cases, with distant metastases to the lungs, bones, liver, and brain being common. Over the past few decades, treatment options for RCC have included surgery, chemotherapy, radiotherapy, cytokine therapy, targeted therapy, and immune checkpoint blockade (ICB). Since the FDA approved nivolumab for clinical treatment in 2015, several immune checkpoint inhibitors have been widely used in the treatment of RCC. However, immune checkpoint inhibitory therapy is not effective in all patients, and its efficacy is influenced by various factors, including individual differences, tumor stage, pathological type, and the immune microenvironment. Given that the expression level of PD-L1 directly affects the efficacy of ICB treatment, and some patients with renal cell carcinoma respond poorly to ICB due to low PD-L1 expression, the treatment of renal cell carcinoma still faces many challenges.

[0003] Therefore, there is an urgent need in this field to find more effective treatment methods and drugs for immune checkpoint inhibition therapy against tumors. Summary of the Invention

[0004] This invention aims to provide a more effective treatment method and drug for immune checkpoint inhibition therapy against tumors. Specifically, it relates to the use of dimethylfumaric acid in promoting immunotherapy for renal cell carcinoma.

[0005] In a first aspect of the invention, a pharmaceutical composition or kit is provided, comprising a therapeutically effective amount of dimethylfumaric acid (DMF), a tumor immunotherapy agent, and a pharmaceutically acceptable carrier.

[0006] In another preferred embodiment, the tumor immunotherapy agent is selected from the group consisting of: PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, and TIM-3 inhibitors.

[0007] In another preferred embodiment, the tumor immunotherapy agent is selected from the group consisting of: PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, TIGIT antibody, and TIM-3 antibody.

[0008] In another preferred embodiment, the tumor immunotherapy agent is selected from the group consisting of CD274, Pembrolizumab, Nivolumab, Atezolizumab, Durvalumab, or pharmaceutically acceptable formulations thereof.

[0009] In another preferred embodiment, the tumor immunotherapy agent is a small molecule inhibitor targeting PD-1, PD-L1, CTLA-4, or a pharmaceutically acceptable salt thereof.

[0010] In another preferred embodiment, the tumor is selected from the group consisting of: renal cell carcinoma, non-small cell lung cancer, melanoma, liver cancer, breast cancer, and colorectal cancer.

[0011] In another preferred embodiment, the renal cell carcinoma is a clear cell renal cell carcinoma.

[0012] In another preferred embodiment, the ratio of DMF to tumor immunotherapy agent in the pharmaceutical composition or kit is 15-20:1 (by mass).

[0013] In another preferred embodiment, the ratio of DMF to tumor immunotherapy agent in the pharmaceutical composition or kit is 18:1 (by mass).

[0014] In another preferred embodiment, the dimethyl fumaric acid is formed by the esterification reaction of the two carboxyl groups (-COOH) of fumaric acid with methanol.

[0015] In another preferred embodiment, the dimethylfumaric acid increases the protein level of PD-L1.

[0016] In another preferred embodiment, the pharmaceutical composition enhances CD8. + The degree of T cell infiltration.

[0017] In another preferred embodiment, the pharmaceutical composition enhances CD8. + The enhancement rate of T cell infiltration is greater than 90%, preferably greater than 95%.

[0018] In another preferred embodiment, the pharmaceutical composition enhances the expression of granzyme B.

[0019] In another preferred embodiment, the pharmaceutical composition enhances the expression of granzyme B by more than 90%, preferably more than 93%.

[0020] In another preferred embodiment, the pharmaceutical composition reduces tumor mass.

[0021] In another preferred embodiment, the pharmaceutical composition reduces tumor mass by an enhancement rate of greater than 60%, preferably greater than 65%.

[0022] In another preferred embodiment, the pharmaceutical composition exhibits an enhancement rate of greater than 60% in reducing tumor volume, more preferably greater than 70%.

[0023] In another preferred embodiment, the enhancement rate is relative to the application of the tumor immunotherapy agent alone in the tumor tissue.

[0024] In another preferred embodiment, the dimethylfumaric acid improves the precision with which tumor immunotherapy agents target tumor cells.

[0025] In another preferred embodiment, the dimethylfumaric acid improves the accuracy of PD-L1 antibody targeting tumor cells.

[0026] In another preferred embodiment, the dimethylfumaric acid improves the accuracy of PD-L1 antibody targeting renal cell carcinoma cells.

[0027] In a second aspect of the invention, there is provided the use of the pharmaceutical composition or kit according to the first aspect for preparing a pharmaceutical composition for treating tumors, said pharmaceutical composition comprising a therapeutically effective amount of dimethylfumaric acid (DMF) and a tumor immunotherapy agent and a pharmaceutically acceptable carrier; preferably, said tumor is renal cell carcinoma.

[0028] In another preferred embodiment, the dimethylfumaric acid and the tumor immunotherapy agent are prepared as the same or different formulations.

[0029] In another preferred embodiment, dimethyl fumaric acid is administered before the administration of the tumor immunotherapy agent.

[0030] In another preferred embodiment, the dimethylfumaric acid and the tumor immunotherapy agent are prepared as different formulations.

[0031] In another preferred embodiment, the tumor immunotherapy agent and dimethylfumaric acid are administered simultaneously.

[0032] In another preferred embodiment, the dimethylfumaric acid and the tumor immunotherapy agent are prepared as the same formulation.

[0033] In another preferred embodiment, the dimethyl fumaric acid is administered at a dose of 20-40 mg / kg.

[0034] In another preferred embodiment, the dimethyl fumaric acid is applied once every 1-3 days.

[0035] In another preferred embodiment, the tumor therapeutic agent is administered at a dose of 90-110 μg per administration.

[0036] In another preferred embodiment, the tumor treatment agent is administered once every 1-5 days.

[0037] In another preferred embodiment, the renal cell carcinoma is a clear cell renal cell carcinoma.

[0038] In another preferred embodiment, the dosage form of the pharmaceutical composition includes tablets, capsules, powders, granules, or oral liquids.

[0039] In a second aspect of the invention, a use of dimethyl fumaric acid (DMF) is provided for the preparation of a sensitizer for tumor immunotherapy agents.

[0040] In another preferred embodiment, the dimethylfumaric acid increases the PD-L1 protein level in tumor cells.

[0041] In another preferred embodiment, the dimethylfumaric acid inhibits T cell expression.

[0042] In another preferred embodiment, the T cells are selected from the group consisting of: CD8 + T cells, CD4 + T cells, regulatory T cells (Tregs), preferably CD8 + T cells.

[0043] In another preferred embodiment, the dimethylfumaric acid has an inhibitory rate of greater than 60% on the expression of T cells.

[0044] In another preferred embodiment, the tumor immunotherapy agent is selected from the group consisting of: PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, and TIM-3 inhibitors.

[0045] In another preferred embodiment, the tumor immunotherapy agent is selected from the group consisting of: PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, TIGIT antibody, and TIM-3 antibody.

[0046] In another preferred embodiment, the tumor immunotherapy agent is selected from the group consisting of CD274, Pembrolizumab, Nivolumab, Atezolizumab, Durvalumab, or pharmaceutically acceptable formulations thereof.

[0047] In another preferred embodiment, the tumor immunotherapy agent is a small molecule inhibitor targeting PD-1, PD-L1, CTLA-4, or a pharmaceutically acceptable salt thereof.

[0048] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0049] Figure 1 The graph shows the effect of DMF on enhancing the protein level of PD-L1 in renal cell carcinoma cells; in which Figure 1 In this context, A represents the time-gradient upregulation of PD-L1 protein levels by DMF. Figure 1 In this context, B represents DMF upregulating PD-L1 protein levels via a concentration gradient.

[0050] Figure 2 This study demonstrates the effect of DMF on PD-L1 expression levels in tumor cells other than renal cell carcinoma.

[0051] Figure 3 The image shows the effect of DMF on promoting the killing of renal cell carcinoma cells by T cells in vitro; among which... Figure 3 In the diagram, A represents surviving tumor cells stained with crystal violet. Figure 3 In this context, B represents the survival rate of tumor cells;

[0052] Figure 4 The graph shows the effect of DMF on enhancing immunotherapy for renal cell carcinoma in mice. The combined use of DMF and PD-L1 antibody promotes the effect of PD-L1 antibody immunotherapy. Figure 4 In the figure, A represents the volume of a mouse renal cell carcinoma tumor. Figure 4 In the figure, B represents the tumor growth curve of renal cell carcinoma in mice. Figure 4 In the figure, C represents the tumor quality statistics of renal cell carcinoma in mice. Figure 4 In this context, D represents CD8 in AGS tumor tissue. + Immunohistochemical staining of T cell infiltration and granzyme B expression. Figure 4 In this context, E represents the area occupied by granzyme in each group. Figure 4 F in the figure represents CD8 in each group. + T cell count. Detailed Implementation

[0053] Through extensive and in-depth research and numerous experimental screenings, the inventors unexpectedly discovered for the first time that dimethylfumaric acid (DMF) can enhance the anti-tumor effects of tumor immunotherapy agents. Specifically, this invention provides a pharmaceutical composition or kit comprising a therapeutically effective amount of dimethylfumaric acid (DMF), a tumor immunotherapy agent, and a pharmaceutically acceptable carrier; wherein the dimethylfumaric acid (DMF) enhances the anti-tumor effects of the tumor immunotherapy agent and CD8 by increasing the protein level of PD-L1. + The anti-tumor immune activity of T cells enhances the efficacy of tumor immunotherapy.

[0054] Furthermore, DMF combined with PD-L1 antibody therapy significantly increased CD8 levels in tumor tissue. +The degree of T cell infiltration and the expression level of granzyme B were affected by the addition of DMF, which increased CD8 expression. + The degree of T cell infiltration and the enhancement rate of granzyme B were greater than 90%, and the enhancement rate of tumor mass and tumor volume reduction was greater than 60%. Therefore, this combination regimen suggests that the combination enhances the tumor therapeutic agent and CD8 through DMF. + The anti-tumor immune activity of T cells enhances the efficacy of tumor immunotherapy, especially DMF, which has a significant enhancing effect on anti-PD-L1 antibody tumor immunotherapy agents. This suggests that DMF has potential clinical application value as a "sensitizer" for ICB therapy. Based on this, the inventors completed this invention.

[0055] Terminology Explanation

[0056] Dimethylfumaric acid

[0057] Dimethyl fumarate (DMF), also known as dimethyl fumarate, is an unsaturated carboxylic acid ester. Its structure is formed by the esterification of the two carboxyl groups (-COOH) of fumarate with methanol, producing two methyl ester (-COOCH3) groups. Its chemical formula is C6H8O4, and its molecular weight is 144.13 g / mol. DMF was initially used to treat psoriasis and has since been widely applied to the treatment of various immune-related diseases, including multiple sclerosis. However, the immunotherapeutic potential of DMF in renal cell carcinoma and its related mechanisms remain unclear.

[0058] In this invention, the inventors unexpectedly discovered that dimethylfumaric acid can increase the expression level of PD-L1 and enhance the therapeutic effect of ICB, suggesting that DMF has potential clinical application value as a "sensitizer" for ICB treatment.

[0059] Immunotherapy for kidney cancer

[0060] Immunotherapy for renal cell carcinoma primarily revolves around immune checkpoint inhibitors, with PD-1 immunotherapy becoming a first-line treatment strategy for metastatic clear cell renal cell carcinoma (ccRCC). PD-1 antibodies (such as nivolumab) restore the tumor-killing function of T cells by blocking the binding of PD-1 on the surface of T cells to PD-L1 on the surface of tumor cells, thereby improving patient survival. In recent years, PD-L1 antibodies (such as atezolizumab) have also been used in renal cell carcinoma immunotherapy, often in combination with other targeted drugs (such as bevacizumab) to enhance the immune response and delay disease progression, significantly improving the treatment prognosis of patients with advanced renal cell carcinoma.

[0061] In this invention, the inventors unexpectedly discovered a new approach to preparing a sensitizer for tumor immunotherapy agents using dimethyl fumaric acid (DMF). By using DMF to inversely increase the protein level of PD-L1 and inhibit the killing power of T cells, the inventors were able to improve the accuracy of PD-L1 antibody targeting tumor cells, thereby significantly inhibiting the growth of tumor cells and improving the anti-tumor effect.

[0062] In addition to PD-1 antibodies (such as nivolumab and pembrolizumab), ligand-inhibiting antibodies can also be used as alternative or combination therapies in PD-1 immunotherapy. These include PD-L1 antibodies (such as atezolizumab, avelumab, and durvalumab) and PD-L2 antibodies currently under investigation. These antibodies enhance the anti-tumor immune response by blocking the PD-1 / PD-L1 or PD-1 / PD-L2 pathways, thereby relieving T-cell suppression. Compared to PD-1 antibodies, PD-L1 antibodies, while blocking PD-1 / PD-L1 signaling, preserve the interaction between PD-1 and PD-L2, potentially exhibiting different immunomodulatory properties. Clinically, they can be selected or combined based on the specific condition to improve efficacy.

[0063] Preferably, the present invention provides a pharmaceutical composition or kit, characterized in that it comprises a therapeutically effective amount of dimethylfumaric acid (DMF), a tumor immunotherapy agent, and a pharmaceutically acceptable carrier.

[0064] The tumor immunotherapy agents are selected from the following group: PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, TIGIT antibody, and TIM-3 antibody.

[0065] Preferably, the tumor immunotherapy agent is selected from the group consisting of CD274, Pembrolizumab, Nivolumab, Atezolizumab, Durvalumab, or pharmaceutically acceptable formulations thereof.

[0066] Preferably, the tumor immunotherapy agent is a small molecule inhibitor targeting PD-1, PD-L1, CTLA-4, or a pharmaceutically acceptable salt thereof.

[0067] Preferably, the tumor is selected from the group consisting of: renal cell carcinoma, non-small cell lung cancer, melanoma, liver cancer, breast cancer, and colorectal cancer.

[0068] Preferably, the renal cell carcinoma is clear cell renal cell carcinoma.

[0069] Preferably, in the pharmaceutical composition or kit, the ratio of DMF to tumor immunotherapy agent is 15-20:1 (by mass).

[0070] Preferably, the ratio of DMF to tumor immunotherapy agent is 18:1 (by mass).

[0071] Preferably, the dimethyl fumaric acid is formed by the esterification reaction of the two carboxyl groups (-COOH) of fumaric acid with methanol.

[0072] Preferably, the dimethylfumaric acid increases the protein level of PD-L1.

[0073] Preferably, the pharmaceutical composition enhances CD8. + The degree of T cell infiltration.

[0074] In another preferred embodiment, the dimethylfumaric acid improves the precision with which tumor immunotherapy agents target tumor cells.

[0075] Preferably, the dimethylfumaric acid improves the accuracy of PD-L1 antibody targeting tumor cells.

[0076] Preferably, the dimethylfumaric acid improves the accuracy of PD-L1 antibody targeting renal cell carcinoma cells.

[0077] In the pharmaceutical compositions or kits of the present invention, dimethylfumaric acid is used in compositions or methods for enhancing the antitumor efficacy of immune checkpoint inhibitors, including the combined application of DMF and PD-L1 antibody to improve the immunotherapy effect of clear cell renal cell carcinoma. Animal experimental results show that in mouse models, compared with the control group or DMF alone, PD-L1 antibody can significantly inhibit tumor growth, and the combined use of DMF and PD-L1 antibody further enhances the antitumor effect, showing a more significant tumor-suppressive effect. Figure 4 This finding suggests that DMF could serve as an adjuvant enhancer for immunotherapy, improving the therapeutic response to immune checkpoint inhibitors such as PD-L1 antibodies.

[0078] It is worth emphasizing that DMF, as a clinically approved drug for the treatment of psoriasis and multiple sclerosis, has well-defined pharmacokinetic characteristics, good safety profile, and low cost. This invention reveals for the first time the novel application prospects of DMF as a "repurposed drug" in the immunotherapy of renal cell carcinoma, which has significant clinical translational value.

[0079] Pharmaceutical Compositions and Administration

[0080] Because the pharmaceutical compositions or combinations thereof of the present invention have excellent therapeutic effects on tumors, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate diseases associated with abnormal activity or expression of type I PRMT.

[0081] In a preferred embodiment, the composition contains 0.1 nM to 50 μM of a first active ingredient, more preferably 0.5 nM to 30 μM, more preferably 1 nM to 10 μM, for example, about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 800 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. The "therapeutic effective dose" refers to: 1-200 mpk of the first active ingredient, preferably 5-100 mpk, more preferably 10-100 mpk, for example, about 20 mpk, 25 mpk, 30 mpk, 35 mpk, 40 mpk, 50 mpk, 55 mpk, 60 mpk, 65 mpk, 70 mpk, 75 mpk, 80 mpk, 85 mpk, or 90 mpk.

[0082] In a preferred embodiment, the composition contains 0.1 nM to 100 μM of a second active ingredient, more preferably 0.1 nM to 50 μM, more preferably 0.1 nM to 20 μM, for example, about 0.3 nM, 0.5 nM, 0.8 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 300 nM, 400 nM, 500 nM, 800 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 15 μM, or 20 μM. The "therapeutic effective dose" refers to: 0.1 to 200 mpk, preferably 0.5 to 150 mpk, more preferably 1 to 100 mpk, for example, about 1 mpk, 5 mpk, 10 mpk, 15 mpk, 20 mpk, 25 mpk, 30 mpk, 35 mpk, 40 mpk, 45 mpk, 50 mpk, 55 mpk, 60 mpk, 65 mpk, 70 mpk, 75 mpk, 80 mpk, 85 mpk, 90 mpk, 95 mpk, or 100 mpk. In a preferred embodiment, the composition contains a third active ingredient of 10 to 1000 μCi / mL, more preferably 50 to 800 μCi / mL, more preferably 100 to 500 μCi / mL, for example, about 100 μCi / mL, 200 μCi / mL, 300 μCi / mL, 400 μCi / mL, or 500 μCi / mL.

[0083] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the first or second active ingredient of the present invention per dose, more preferably, 5-500 mg of the first or second active ingredient of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0084] In this invention, the first and second active ingredients can be administered co-administered or separately. When administered co-administered, the first and second active ingredients are prepared into the same pharmaceutical dosage form for use, while when administered separately, the first and second active ingredients can be given to the desired recipient simultaneously or separately.

[0085] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0086] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

[0087] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in the dosage forms of capsules, tablets, and pills.

[0088] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0089] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0090] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0091] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0092] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0093] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0094] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds. In some preferred embodiments, the compounds of this invention can be administered together with other small molecule compounds to form a PROTAC, or together with other large molecule compounds such as monoclonal antibodies to form an ADC.

[0095] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 5–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0096] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0097] 1. This invention provides a novel approach for preparing a sensitizer for tumor immunotherapy agents using dimethylfumaric acid (DMF). The dimethylfumaric acid increases the protein level of PD-L1, inhibits the tumor-killing ability of T cells, and improves the accuracy of PD-L1 antibody targeting tumor cells.

[0098] 2. The pharmaceutical composition of the present invention comprises a therapeutically effective amount of dimethylfumaric acid (DMF), a tumor immunotherapy agent, and a pharmaceutically acceptable carrier. The pharmaceutical composition can significantly inhibit the growth of tumor cells and improve the anti-tumor effect by improving the precision of PD-L1 antibody targeting tumor cells.

[0099] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0100] The PD-L1 antibody described in this example is InVivoMAb anti-mouse PD-L1 (CD274), a commonly used PD-L1 antibody in the art. Those skilled in the art may also select other PD-L1 antibodies to use in combination with DMF according to clinical or experimental needs.

[0101] Example 1: DMF enhances PD-L1 protein levels in renal cell carcinoma cells.

[0102] In this embodiment, human renal cell carcinoma RCC10 and RCC4 cells were cultured in a cell culture medium containing DMF, and the protein level of PD-L1 in the cells was detected after culture.

[0103] Human renal cell carcinoma RCC10 and RCC4 cells were treated with cell culture medium containing 50 μM DMF at time gradients of 0 h, 3 h, 6 h, 12 h, and 24 h. The protein level of PD-L1 in the cells was detected by Western blot. The results are as follows: Figure 1 As shown in A.

[0104] Renal cell carcinoma RCC10 and RCC4 cells were treated with DMF-containing cell culture medium and DMF concentration gradients of 0 μM, 10 μM, 25 μM, and 50 μM. After 12 hours, the protein level of PD-L1 in the cells was detected by Western blot. The results are as follows: Figure 1 As shown in B.

[0105] The results showed that DMF can enhance the protein expression level of PD-L1 in renal clear cell carcinoma RCC10 and RCC4 cells, and this regulatory effect is time- and concentration-dependent, with the two cell lines showing a highly consistent response pattern.

[0106] Example 2: DMF's effect on PD-L1 protein levels in other types of tumor cells

[0107] In this embodiment, other types of tumor cells were cultured in a cell culture medium containing DMF. These other types of tumor cells included human non-small cell lung cancer H1299, human melanoma A375, human liver cancer HCCLM3, human breast cancer MDA-MB-231, mouse colorectal cancer CT26, and mouse breast cancer 4T1 tumor cell lines. After culture, the protein level of PD-L1 in each type of cell was detected.

[0108] Western blot analysis was performed on the PD-L1 protein levels of human non-small cell lung cancer H1299, human melanoma A375, human liver cancer HCCLM3, human breast cancer MDA-MB-231, mouse colorectal cancer CT26, and mouse breast cancer 4T1 tumor cell lines after treatment with DMF (50 μM, 12 h). Figure 2 As shown.

[0109] The results showed that DMF did not cause significant changes in PD-L1 protein expression in tumor cell lines other than renal cell carcinoma.

[0110] Example 3: DMF inhibits the killing of renal cell carcinoma cells by T cells in vitro.

[0111] a) In vitro culture and activation of CD8+ T cells. 10 ml of fresh blood was obtained from healthy adult volunteers, and human peripheral blood mononuclear cells (PBMCs) were isolated. PBMCs were cultured in ImmunoCult-XF T cell expansion medium containing IL-2 (10 ng / mL). For the first 3 days of culture, PBMCs were activated using ImmunoCult Human CD3 / CD28 / CD2 T cell activator (25 μg / ml). Activated human CD8+ T cells were obtained after 7 days of culture.

[0112] b) DMF treatment of tumor cells. RCC4 tumor cells were divided into two groups: the control group was cultured in standard DMEM medium, and the treatment group was cultured in DMEM medium containing 50 μM DMF. After 12 hours of culture, both groups of cells were digested with trypsin and counted, then evenly seeded into 24-well plates, with 2 wells in each group.

[0113] c) Co-culture of CD8+ T cells and tumor cells. The activated CD8+ T cells described above... + T cells were added to a tumor cell culture plate at a ratio of 1:10 (tumor cells:T cells) and co-cultured in DMEM / F12 medium containing CD3 antibody (100 ng / mL) and IL-2 (10 ng / mL).

[0114] d) Detection of tumor cell viability. After co-culturing CD8+ T cells with tumor cells for 48 hours, the liquid in the culture plate was aspirated, and cell debris was washed away with PBS. The surviving tumor cells in the cell culture plate were stained with crystal violet, then destained with 33% (m / v) acetic acid solution. Finally, the absorbance of the destained solution at 570 nm was measured. Results are as follows: Figure 3 As shown in Table 1.

[0115] From Table 1 and Figure 3As can be seen, adding DMF alone to tumor cells (Table 1, No. 3) did not significantly change the tumor cell survival rate, indicating that DMF itself has no inhibitory effect on tumors. Adding T cells alone to tumor cells (Table 1, No. 2) decreased the tumor cell survival rate by approximately 78.2%, while pretreatment of T cells with DMF (Table 1, No. 4) increased the tumor cell survival rate to 85.8%, meaning that DMF inhibited the tumor cell killing effect of T cells by approximately 62%. Therefore, these results indicate that DMF pretreatment of T cells significantly inhibited the killing effect of T cells on RCC4 cells, suggesting that DMF may affect the anti-tumor activity of T cells by regulating immune checkpoint molecules or the tumor immune microenvironment.

[0116] Table 1. The killing effect of DMF on T cells against tumors.

[0117]

[0118] Example 4: Combined use of DMF and PD-L1 antibodies enhances the efficacy of immunotherapy for renal cell carcinoma.

[0119] In this embodiment, DMF and PD-L1 antibodies were jointly administered to tumor cells to study their therapeutic effect on tumors. The specific experimental steps are as follows:

[0120] a) Construction of a mouse renal cell carcinoma model. 1×10 7 When 4T1 mouse renal cell carcinoma cells are injected subcutaneously into female BALB / c mice, solid renal cell carcinoma tumors can grow after about 7 days.

[0121] b) The experimental mice were divided into four groups: control group, DMF-only group, PD-L1 antibody-only group, and DMF and PD-L1 antibody combination group;

[0122] c) Administration method: Based on the experimental objectives, this study set up four experimental groups, namely:

[0123] Control group, PD-L1 antibody treatment group, DMF treatment group, and PD-L1 antibody combined with DMF treatment group;

[0124] PD-L1 antibody treatment group administration method: Each mouse was injected intraperitoneally with 100 μg of PD-L1 antibody every three days;

[0125] DMF treatment group administration method: Each mouse was administered 30 mg / kg by gavage daily;

[0126] The administration method for the combined treatment group of DMF and PD-L1 antibody was as follows: each mouse was administered DMF by gavage at a dose of 30 mg / kg per day and PD-L1 antibody by intraperitoneal injection every three days;

[0127] Throughout the treatment course, DMF was administered 21 times and PD-L1 antibody was administered 7 times.

[0128] d) Tumor volume measurement. Starting from the start of tumor inoculation, the length and width of the tumor were measured every three days using calipers, and the volume was calculated.

[0129] e) Graphs showing tumor volume in tumor-bearing mice, tumor growth curves, tumor mass statistics, and immunohistochemical staining of tumor tissue, such as... Figure 4 As shown in Table 2, the changes in tumor weight are shown in Table 3, the changes in tumor volume are shown in Table 4, the number of CD8+ T cells in AGS tumor tissue is shown in Table 5, and the expression of granzyme B in AGS tumor tissue is shown in Table 6.

[0130] Table 2 Tumor Quality

[0131]

[0132] Table 3 Tumor Volume

[0133]

[0134]

[0135]

[0136] Table 4CD8 + T cell count

[0137]

[0138] Table 5. Area occupied by granzyme B in AGS tumor tissue

[0139]

[0140] Table 6. Enhancement effect of DMF on anti-PD-L1 antibody tumor immunotherapy agents

[0141]

[0142]

[0143] Note: Enhancement rate calculation method:

[0144] Depend on Figure 4 As shown in Tables 2-3, tumor volume and mass increased after DMF treatment alone, a phenomenon consistent with in vitro experimental results, suggesting that DMF can promote tumor immune escape. Furthermore, in the group treated with anti-PD-L1 antibody alone, PD-L1 antibody significantly inhibited tumor growth and reduced tumor weight.

[0145] In this invention, further IHC analysis was performed to show ( Figure 4 (and Tables 4 and 5) CD8 levels in tumor tissue after DMF treatment alone + The number of T cells was reduced, and granzyme B was also reduced. In the group treated with anti-PD-L1 antibody alone, PD-L1 antibody increased CD8+. + T cell count and granzyme B expression.

[0146] Surprisingly, the combination therapy of DMF and PD-L1 antibody showed the best anti-tumor effect. (See Table 6 and...) Figure 4 As can be seen, compared with PD-L1 antibody therapy, DMF combined with PD-L1 antibody therapy significantly increased CD8 levels in tumor tissue. + The degree of T cell infiltration and the expression level of granzyme B, and the addition of DMF increased CD8. + The T cell infiltration rate and granzyme B enhancement rate were 96.68% and 93.50%, respectively, while the reduction in tumor mass and volume was enhanced by 66.87% and 75.03%, respectively. Therefore, these results suggest that this combined regimen enhances the effects of tumor therapeutic agents and CD8 by increasing PD-L1 protein levels through DMF. + The anti-tumor immune activity of T cells enhances the efficacy of tumor immunotherapy, especially DMF, which has a significant enhancing effect on PD-L1 antibody tumor immunotherapy agents.

[0147] In summary, the combination therapy of DMF and tumor therapeutic agents can effectively enhance the sensitivity of tumor immunotherapy agents. In particular, the combination therapy of DMF and PD-L1 antibody significantly enhances the accuracy of tumor immunotherapy agents, enabling them to accurately target tumor tissue and significantly inhibit the growth of renal cell carcinoma xenografts. This suggests that DMF has potential clinical application value as a "sensitizer" for ICB therapy.

[0148] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A pharmaceutical composition or kit, characterized in that, It contains therapeutically effective amounts of dimethylfumaric acid (DMF) and tumor immunotherapy agents and pharmaceutically acceptable carriers.

2. The pharmaceutical composition or kit according to claim 1, characterized in that, The tumor immunotherapy agents are selected from the following group: PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, and TIM-3 inhibitors.

3. The pharmaceutical composition or kit according to claim 1, characterized in that, The tumor immunotherapy agents are selected from the following group: PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, TIGIT antibody, and TIM-3 antibody.

4. The pharmaceutical composition or kit according to claim 1, characterized in that, The tumor immunotherapy agent is selected from the group consisting of CD274, Pembrolizumab, Nivolumab, Atezolizumab, Durvalumab, or pharmaceutically acceptable formulations thereof.

5. The pharmaceutical composition or kit according to claim 1, characterized in that, The tumor immunotherapy agent is a small molecule inhibitor targeting PD-1, PD-L1, CTLA-4, or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition or kit according to claim 1, characterized in that, The tumors are selected from the following group: renal cell carcinoma, non-small cell lung cancer, melanoma, liver cancer, breast cancer, and colorectal cancer.

7. The pharmaceutical composition or kit according to claim 1, characterized in that, In the pharmaceutical composition or kit described above, the ratio of DMF to tumor immunotherapy agent is 15-20:1 (by mass).

8. Use of the pharmaceutical composition or kit according to claim 1, characterized in that, A pharmaceutical composition for treating tumors, the pharmaceutical composition comprising a therapeutically effective amount of dimethylfumaric acid (DMF) and a tumor immunotherapy agent and a pharmaceutically acceptable carrier; preferably, the tumor is renal cell carcinoma.

9. In the use according to claim 8, the dimethylfumaric acid and the tumor immunotherapy agent are prepared as the same or different formulations.

10. A use of dimethylfumaric acid (DMF), characterized in that, Sensitizers used in the preparation of tumor immunotherapy agents.

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