A guanidyl polymer capable of competitively binding to a PD-L1 domain of a cell membrane, and a preparation method and application thereof

Abstract

The application provides a guanidyl polymer capable of competitively binding to a PD-L1 domain of a cell membrane, and a preparation method and application thereof, the guanidyl polymer has a flexible carbon main chain and a rigid side chain containing a guanidyl group, and can effectively reduce the expression level of PD-L1 on the membrane surface of tumor cells and immune cells. Specifically, the guanidyl polymer can effectively target the intracellular domain of PD-L1, simulate the key basic amino acid functional region thereof, competitively bind to the intramembrane acid phospholipid of the cell membrane through the side chain guanidyl group, interfere with the membrane anchoring stability of PD-L1-ICD, and thus effectively reduce the PD-L1 level on the membrane surface of tumor cells and immune cells.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to a guanidine polymer that competitively binds to the PD-L1 domain of the cell membrane, its preparation method, and its application. Background Technology

[0002] In recent years, immunotherapy, represented by immune checkpoint blockade, has made groundbreaking progress and plays an important role in the clinical treatment of various types of malignant tumors, significantly prolonging the survival of cancer patients. In particular, immune checkpoint blockade therapies targeting adaptive immune cells, such as antibodies targeting programmed death receptor-1 ligand (PD-L1), have achieved great clinical success in treating various solid tumors and hematological malignancies, including melanoma, non-small cell lung cancer, and high-level microsatellite instability colorectal cancer.

[0003] However, clinically approved antibody drugs targeting PD-L1 primarily target its extracellular domain, i.e., by competitively blocking the binding of PD-1 to PD-L1 outside the cell, thereby relieving the inhibition of T cell activation. Although these conventional antibodies can affect the binding function of PD-L1 on the cell membrane, the PD-L1 protein itself still persists on the cell membrane, and its potential inhibitory effect on T cells cannot be completely eliminated. Summary of the Invention

[0004] To overcome at least one of the problems existing in the prior art, one object of the present invention is to provide a guanidine polymer. A second object of the present invention is to provide a method for preparing the above-mentioned guanidine polymer. A third object of the present invention is to provide a pharmaceutical composition. A fourth object of the present invention is to provide an application of the above-mentioned guanidine polymer. A fifth object of the present invention is to provide another application of the above-mentioned guanidine polymer.

[0005] Studies have shown that the intracellular domain (ICD) of PD-L1 regulates PD-L1 protein stability and mediates intracellular signal transduction through multiple pathways. Novel intervention strategies targeting the PD-L1-ICD hold promise for reducing PD-L1 levels on the cell membrane surface and completely eliminating its immunosuppressive function against T lymphocytes by interfering with PD-L1 protein stability and inducing PD-L1 protein degradation. Therefore, PD-L1-ICD shows promise as a potential target for reducing PD-L1 levels on the cell membrane surface. However, since the PD-L1-ICD is located inside the cell, traditional antibody molecules struggle to penetrate the cell membrane and reach the cytoplasm to exert their effects. There is an urgent need to develop molecular tools that can efficiently enter cells and specifically target the PD-L1-ICD.

[0006] The study found that within this structural domain, PD-L1-ICD 260-265Employing an α-helical cationic structure, the interaction between three key arginine residues on PD-L1 and the acidic phospholipids of the inner membrane is crucial for PD-L1 membrane anchoring. Replacing these residues with other cationic amino acids significantly weakens its membrane anchoring ability. Furthermore, PD-L1-ICD... 270-281 The abundant lysine residues in the region also participate in the membrane anchoring of the ICD and have been shown to be closely related to the ubiquitination and degradation of PD-L1. Therefore, to simulate PD-L1-ICD... 260-281 This invention innovatively proposes a biostable cationic polymer, polymethyl guanidine (PMet), with a flexible carbon backbone and rigid side chains containing guanidine groups, which can effectively reduce the expression level of PD-L1 on the surface of tumor cells and immune cells.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a guanidine polymer, the structural formula of which is shown in Formula A below:

[0008] Formula A in, The replacement rate is greater than 90%.

[0009] The substitution rate refers to the proportion of secondary amines on linear polyethyleneimine (PEI) that undergo substitution reactions, i.e., substitution rate = reacted secondary amine / total secondary amines × 100%.

[0010] Preferably, the raw materials for preparing the guanidine polymer include linear polyethyleneimine and dicyandiamide.

[0011] More preferably, the linear polyethyleneimine is a low / medium molecular weight linear polyethyleneimine.

[0012] More preferably, the linear polyethyleneimine has a molecular weight of 1000-50000.

[0013] Preferably, the substitution rate is greater than 95%.

[0014] More preferably, the substitution rate is greater than 98%.

[0015] A second aspect of the present invention provides a method for preparing the guanidine polymer described in the first aspect, comprising the following steps: The guanidine polymer is prepared by reacting linear polyethyleneimine with dicyandiamide.

[0016] Preferably, the mass ratio of the linear polyethyleneimine to dicyandiamide is 1:(10-20).

[0017] More preferably, the mass ratio of the linear polyethyleneimine to dicyandiamide is any value or a range of any two values ​​from 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20.

[0018] Preferably, the reaction temperature is 90-120°C.

[0019] More preferably, the reaction temperature is any value or a range of any two values ​​from 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120°C.

[0020] Preferably, the reaction time is 20-35 h.

[0021] More preferably, the reaction time is any value or a range of any two values ​​from 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 h.

[0022] Preferably, the method further includes the following steps: dissolving linear polyethyleneimine in hydrochloric acid solution, and then adding dicyandiamide to react.

[0023] More preferably, the concentration of the hydrochloric acid solution is 2-3 mol / L.

[0024] A third aspect of the present invention provides a pharmaceutical composition comprising the guanidine polymer described in the first aspect.

[0025] Preferably, it also includes one or more pharmaceutically acceptable excipients.

[0026] More preferably, the pharmaceutically acceptable excipient refers to a drug carrier conventional in the pharmaceutical field, including at least one of fillers, binders, disintegrants, solubilizers, suspending agents, wetting agents, pigments, fragrances, solvents, surfactants, or flavoring agents.

[0027] Substances that can be used as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers; aluminum; aluminum stearate; lecithin; serum proteins, such as human serum albumin; buffering substances such as phosphates; glycine; sorbic acid; potassium sorbate; mixtures of partial glycerides of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts; colloidal silica; magnesium trisilicate; polyvinylpyrrolidone; polyacrylates; waxes; polyethylene-polyoxypropylene-blocking polymers; lanolin; sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives. Biological agents such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic salts; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic and suitable lubricants such as sodium lauryl sulfate and magnesium stearate; colorants; release agents; preservatives and antioxidants.

[0028] Preferably, the dosage form of the composition includes any one of oral formulations, injectable formulations, respiratory formulations, and transdermal formulations.

[0029] More preferably, the dosage forms of oral preparations include granules, tablets, powders, capsules, oral solutions, oral suspensions, dry suspensions, oral emulsions, gels, oral liquids, emulsions, emulsions, colloidal solutions, mixtures, tinctures, drops, suspension drops, syrups, sustained-release tablets, sustained-release coated tablets, controlled-release tablets, sustained-release capsules, and controlled-release capsules.

[0030] More preferably, the dosage form of the injectable preparation includes injections, injection solutions, solutions for injection, injection solutions for intravenous infusion, suspensions for injection, sterile powders for injection, intravenous injections, emulsions for injection, emulsion injections, powder injections, injections, sterile powder injections, lyophilized powder injections, and concentrated solutions for injection.

[0031] The fourth aspect of the present invention provides the use of the guanidine polymer described in the first aspect, or the pharmaceutical composition described in the third aspect, in the preparation of a medicament for treating cancer.

[0032] Preferably, the cancer includes colorectal cancer, lung cancer, melanoma, breast cancer, liver cancer, or renal cell carcinoma.

[0033] Preferably, the drug is used to reduce the level of PD-L1 on the surface of tumor cell membranes.

[0034] Preferably, the drug is used to reduce the level of PD-L1 on the surface of macrophage membranes.

[0035] Preferably, the cancer treatment drug comprises a guanidine polymer in combination with one or more other therapeutic agents. Examples include chemotherapy drugs (such as platinum-based drugs, taxanes, antimetabolites, anthracyclines, etc.), targeted therapies (such as EGFR inhibitors, ALK inhibitors, PARP inhibitors, CDK4 / 6 inhibitors, etc.), immunotherapies (such as PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, etc.), endocrine therapies, anti-angiogenic drugs, antibody-drug conjugates, or any combination thereof.

[0036] The fifth aspect of the present invention provides the use of the guanidine polymer described in the first aspect in the preparation of PD-L1 targeted inhibitors.

[0037] Preferably, the PD-L1-targeting inhibitor is an inhibitor that targets the intracellular domain of PD-L1.

[0038] More preferably, the PD-L1-targeting inhibitor targets the intracellular domain of PD-L1, mimicking its key basic amino acid functional regions, and reduces the PD-L1 level on the surface of tumor cells and / or immune cells by competitively inhibiting PD-L1 membrane anchoring.

[0039] More preferably, the PD-L1-targeting inhibitor is used to mimic PD-L1-ICD. 260-281 Sequence structure rich in basic amino acids (arginine, lysine).

[0040] The beneficial effects of this invention are: This invention provides a guanidine polymer that competitively binds to the PD-L1 domain of the cell membrane. This guanidine polymer has a flexible carbon backbone and rigid side chains containing guanidine groups, effectively reducing PD-L1 expression levels on the surface of tumor cells and immune cells. Specifically, this guanidine polymer can mimic PD-L1-ICD. 260-281 The sequence structure, rich in basic amino acids (arginine, lysine), interferes with the membrane anchoring stability of PD-L1-ICD by competitively binding its side-chain guanidine groups to acidic phospholipids on the inner side of the cell membrane. This reduces the PD-L1 level on the surface of tumor cells (including but not limited to colorectal cancer, lung cancer, melanoma, breast cancer, liver cancer, renal cell carcinoma, etc.) and immune cells (including but not limited to macrophages, dendritic cells, etc.) at the source, thus completely destroying their immunosuppressive function. Attached Figure Description

[0041] Figure 1 The image shows the 1H-NMR spectrum of PMet in D2O. Figure 2The interaction between PD-L1 and PMet and the lipid membrane (Mem) is shown (blue surface represents POPG, white surface represents POPC). Figure 3 PMet competes with PD-L1 for binding to the lipid membrane (blue surface represents POPG, white surface represents POPC, and yellow surface represents PMet). Figure 4 The effects of different Met concentrations or molecular weights of cationic polymers (PEI, PMet) on PD-L1 expression on the surface of M1 macrophage membranes; Figure 5 Flow cytometry analysis (a) and quantitative results (b) of PD-L1 on the surface of tumor cell membranes. Detailed Implementation

[0042] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0043] Example 1 This embodiment provides a guanidine polymer (PMet), the specific synthesis method of which is as follows: 0.1 g of linear polyethyleneimine (PEI, with number-average molecular weights of 2,500, 4,000, or 10,000 g / mol, denoted as PEI-L, PEI-M, and PEI-H, respectively) was dissolved in a round-bottom flask containing 5 mL of 2.5 M hydrochloric acid aqueous solution. 1.2 g of dicyandiamide (DCD) was added, and the mixture was heated in an oil bath to 100 °C, refluxed, and reacted for 24 h. After the reaction was complete, the mixture was dialyzed for two days using a 1 kDa dialysis bag. After freeze-drying, the solution was prepared with sterile, enzyme-free water and stored at 4 °C.

[0044] To verify the successful synthesis of PMet, the reaction products were subjected to 1H NMR spectroscopy (H2O) using deuterated water (D2O) as a solvent. 1 -NMR) analysis. Results are as follows: Figure 1 As shown, the synthesized PMet structure is correct, and the substitution rate of dicyandiamide can be calculated to be close to 98.8% based on the peak areas of 2.71-2.82 ppm and 2.86-4.21 ppm.

[0045] Molecular dynamics simulation The interaction between PMet and the lipid membrane (Mem) composed of 1-palmitoyl-2-oleo-sn-glycerol-3-phosphatidylglycerol (POPG) and 1-palmitoyl-2-oleo-sn-glycerol-3-phosphatidylcholine (POPC) was studied using molecular dynamics simulations. The results are as follows: Figure 2 As shown, PMet and PD-L1 exhibit highly similar membrane binding behaviors.

[0046] Subsequently, molecular dynamics simulations were used to investigate the competitive interaction between PMet and Mem bound to PD-L1. The results are as follows: Figure 3 As shown, before 174 ns, although the PMet molecule was completely embedded in the phospholipid bilayer, the PD-L1-ICD domain was still encapsulated by acidic phospholipids due to the electrostatic bond between the guanidinyl side chain of R260 in PD-L1-ICD and the phosphate group of POPG299 in the lipid membrane. At 174 ns, the phosphate group of POPG299 electrostatically bonded to the PMet side chain, while the binding to R260 of PD-L1-ICD disappeared. After 174 ns, the binding of POPG299 to PMet indicates that PMet can competitively replace the interaction between the guanidinyl group of PD-L1-ICD and the acidic phospholipids.

[0047] Biological Experiment Test Example 1 Using macrophages as a representative of immune cells, we tested the downregulation effect of PMet on PD-L1 levels on the cell membrane surface.

[0048] 1. Extraction of mouse bone marrow-derived macrophages (BMDM) Healthy BALB / c mice were euthanized by cervical dislocation, and their femurs and tibias were harvested. The epiphyses at both ends were removed with scissors, and the bone marrow was washed into centrifuge tubes with DMEM complete medium. The collected bone marrow cell suspension was passed through a 200-mesh (70 μm) nylon sieve and centrifuged at 1200 rpm for 5 min. The supernatant was discarded, and erythrocyte lysis buffer was added to lyse the erythrocytes. The suspension was then centrifuged at 500 g for 5 min at 4°C, and the supernatant was discarded. The cells were washed twice with PBS. The washed bone marrow cell pellet was resuspended in DMEM complete medium containing 20 ng / mL M-CSF. The cells were seeded into culture dishes and cultured at 37°C in an incubator containing 5% CO2, marked as day 0. On day 2, the culture dish was gently shaken, and half the volume of the supernatant was removed. An equal volume of medium was added, and the cells were cultured until day 4. The entire medium was then replaced, and the culture was continued until day 6, at which point the cells were cultured to obtain BMDM.

[0049] 2. The downregulation effect of different molecular weight PMet on PD-L1 levels on macrophage membrane surface The extracted BMDM was inoculated into 6-well plates (2×10⁻⁶). 5M2 or M1 macrophages were induced by culturing cells in complete medium containing IL-4 (20 ng / mL) or LPS (100 ng / mL) for 24 hours in wells. M1 macrophages were then cultured for 48 hours in complete medium containing different concentrations of metformin (Met, 0.5 mM, 1 mM, 2 mM, denoted as Met-l, Met-m, Met-h), 18.75 μg / mL of PMet (the three molecular weights prepared above, denoted as PMet-L, PMet-M, PMet-H), and 18.75 μg / mL of PEI-L, PEI-M, and PEI-H. After culturing, the culture medium was aspirated, the cells were washed with PBS, scraped off with a cell scraper, collected in EP tubes, centrifuged at 500 g for 5 min, and the supernatant was removed. The cells were resuspended in flow cytometry staining buffer, anti-CD16 / 32 antibody was added, and the cells were blocked at 4°C for 10 min. Add APC-labeled anti-PD-L1 antibody and PE-labeled anti-F4 / 80 antibody, and incubate at 4°C in the dark for 1 h. Centrifuge at 500 g for 5 min, discard the supernatant and wash the precipitate. Resuspend the cells in flow cytometry staining buffer and detect the PD-L1 level on the cell surface by flow cytometry.

[0050] The flow cytometry quantification results are represented by mean fluorescence intensity (MFI), such as Figure 4As shown, compared with M2 macrophages, M1 macrophages significantly upregulated membrane PD-L1 expression. A low concentration of 0.5 mM Met (corresponding to a concentration of 82.8 µg / mL, i.e., Met-1) further promoted M1 polarization, leading to a further increase in PD-L1 levels (relative expression level of 146.79 ± 7.94%). However, when the Met concentration increased to 1 mM and 2 mM (corresponding to concentrations of 165.6 µg / mL and 331.2 µg / mL, i.e., Met-m and Met-h), its inhibitory effect on PD-L1 partially offset the polarization-driven increase in PD-L1, resulting in PD-L1 levels comparable to those of M1 macrophages (relative expression levels of 99.25 ± 5.10% and 90.62 ± 1.49%, respectively). In contrast, at a relatively low concentration of 18.75 μg / mL, medium and high molecular weight PMet (PMet-M and PMet-H) effectively reduced PD-L1 levels on the surface of M1 macrophages (relative expression levels of 46.05±4.71% and 26.71±0.84%, respectively), even lower than those in M2 macrophages. Furthermore, the downregulation of surface PD-L1 by PMet showed a clear molecular weight dependence; the higher the molecular weight of PMet used, the lower the PD-L1 level on the macrophage membrane surface. Conversely, the corresponding molecular weight PEI precursor had no significant effect on PD-L1 levels at a concentration of 18.75 μg / mL. These results indicate that PMet, compared to Met, can effectively reduce PD-L1 on the macrophage membrane surface at lower concentrations, while PEI does not have this function.

[0051] Biological Experiment Test Example 2 Using the microsatellite-stabilized mouse colorectal cancer CT26 cell line as a representative tumor cell line, we tested the downregulation effect of PMet on the level of PD-L1 on the cell membrane surface.

[0052] CT26 cells were seeded in 6-well plates (2 × 10⁻⁶ cells per well). 5 CT26 cells were cultured for 48 h in complete medium containing 18.75 μg / mL PMet-M and 18.75 μg / mL PEI-M, respectively, in wells. After culturing, the culture medium was aspirated, the cells were washed with PBS, digested with trypsin, and collected in EP tubes. The cells were centrifuged at 500 g for 5 min, and the supernatant was removed. The cells were resuspended in flow cytometry staining buffer, and anti-CD16 / 32 antibody was added and the cells were blocked at 4 °C for 10 min. APC-labeled anti-PD-L1 antibody was added, and the cells were incubated at 4 °C in the dark for 1 h. After centrifugation at 500 g for 5 min, the supernatant was discarded, the precipitate was washed, and the cells were resuspended in flow cytometry staining buffer. The PD-L1 level on the cell surface was detected by flow cytometry.

[0053] The results are as follows Figure 5 As shown, compared with the Control group, the PEI-M administration group had no effect on the PD-L1 level on the membrane surface (relative expression level of 103.69±3.42%), while the PMet-M administration group was able to downregulate the surface PD-L1 level (relative expression level of 73.29±0.96%).

[0054] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A guanidine polymer, characterized in that, The structural formula of the guanidine polymer is shown in Formula A below: Formula A in, The replacement rate is greater than 90%.

2. The method for preparing the guanidine polymer according to claim 1, characterized in that, Includes the following steps: The guanidine polymer is prepared by reacting linear polyethyleneimine with dicyandiamide.

3. The method for preparing the guanidine polymer according to claim 2, characterized in that, The mass ratio of the linear polyethyleneimine to dicyandiamide is 1:(10-20).

4. The method for preparing the guanidine polymer according to claim 2, characterized in that, The reaction temperature is 90-120℃.

5. A pharmaceutical composition, characterized in that, Includes the guanidine polymer of claim 1.

6. The use of the guanidine polymer of claim 1, or the pharmaceutical composition of claim 5, in the preparation of a medicament for treating cancer.

7. The application according to claim 6, characterized in that, The cancers mentioned include colorectal cancer, lung cancer, melanoma, breast cancer, liver cancer, or renal cell carcinoma.

8. The application according to claim 6, characterized in that, The drug is used to reduce the level of PD-L1 on the surface of tumor cell membranes.

9. The use of the guanidine polymer of claim 1 in the preparation of PD-L1 targeted inhibitors.

10. The application according to claim 9, characterized in that, The PD-L1-targeting inhibitor is an inhibitor that targets the intracellular domain of PD-L1.

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