Preparation method of multifunctional immune agonist for reprogramming tumor-associated macrophages

Abstract

The invention discloses a preparation method of a multifunctional immune agonist for reprogramming tumor-associated macrophages. According to the invention, a precise preparation process based on a solid-phase carrier is developed by taking the siglec-10 receptor epitope as a target and utilizing a disulfide bond as a bridge. The method comprises the following steps: connecting a target to the surface of a thiolated ferroferric oxide magnetic sphere through cysteine modification to construct a solid-phase carrier; synthesizing an amino acid derivative, and forming a functional polymer layer on the surface of the solid-phase carrier through polymerization reaction; and a mercaptan reducing agent is adopted to wash off the target, and the polymer layer with the specific recognition cavity is reserved. The immune agonist can target a siglec-10 receptor, anchors macrophages by identifying a cavity, and gathers to a tumor site under the action of a magnetic field to enhance immune phagocytosis; meanwhile, polarization of macrophages is induced, and the anti-tumor immune function of the macrophages is activated. The agonist is an innovative medicine aiming at tumor-related macrophages, the anti-tumor effect is improved, and the influence on normal tissues is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a preparation method of a multifunctional immune agonist for reprogramming tumor-associated macrophages. Background Art

[0002] Macrophages are an important part of the immune system and exhibit significant phenotypic diversity. In the tumor microenvironment, tumor tissues attract resident macrophages and circulating monocytes, making macrophages the main immune cell population. Macrophages are generally divided into pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. In the tumor microenvironment, especially tumor-associated macrophages (TAMs), tend to adopt an anti-inflammatory M2-like phenotype under various stimuli, which is characterized by the upregulation of cytokines, proteases, and immunosuppressive molecules, thus promoting tumor progression, metastasis, invasion, and resistance to cancer treatment. In multiple cancer types, an increase in TAM infiltration is usually associated with poor patient prognosis. Therefore, TAMs have become a promising target in cancer treatment.

[0003] Currently, researchers have proposed various strategies for tumor-associated macrophages (TAMs) in cancer treatment. These strategies mainly include regulating the migration of monocytes to the tumor site by targeting the signal pathways that regulate macrophage recruitment. In addition, strategies for depleting TAMs and other related methods are constantly being developed and optimized. However, these strategies often neglect the inherent immune function of macrophages. In response, reprogramming of TAMs has become a strategy, mainly achieved by activating the pro-inflammatory signal pathway in TAMs or enhancing their phagocytic activity against tumor cells. Biomolecules such as Toll-like receptor agonists and cytokines have been widely used in the repolarization of TAMs.

[0004] Sialic acid-binding Ig-like lectin 10 (siglec-10) is an inhibitory receptor widely expressed in macrophages and plays a crucial role in immune regulation. Its high-affinity binding to the tumor biomarker small cell lung cancer cluster 4 antigen (CD24) (mainly dependent on sialylation) highlights the status of CD24 as the main ligand of siglec-10. The interaction between CD24 on tumor cells and siglec-10 on macrophages initiates an inhibitory signaling cascade, mainly mediated by Src homology 2 domain-containing phosphatase SHP-1 and / or SHP-2. These phosphatases are associated with immunoreceptor tyrosine-based motifs (ITIMs) or ITIM-like motifs and the cytoplasmic tail of siglec-10, effectively hindering the Toll-like receptor-mediated inflammatory response and the cytoskeletal rearrangement necessary for macrophage-mediated phagocytosis, preventing the recognition of tumor cells by macrophages, and promoting the immune escape of tumor cells. Notably, blocking the CD24 / siglec-10 signaling pathway has shown significant efficacy in achieving effective tumor suppression. Summary of the Invention

[0005] The object of the present invention is to provide a preparation method of a multifunctional immune agonist for reprogramming tumor-associated macrophages. Taking the siglec-10 receptor epitope as the target and disulfide bonds as the "bridge", the present invention innovatively develops a precise preparation process based on a solid-phase carrier. Specifically, first, the target is connected to the surface of mercapto-functionalized magnetite (Fe3O4) magnetic beads through cysteine (C) modified at the end to construct a solid-phase carrier. Subsequently, derivatives of phenylalanine and tyrosine are designed and synthesized based on the epitope peptide, and a functional polymer layer is formed on the surface of the solid-phase carrier through a polymerization reaction. Finally, thiol-based reducing agents such as glutathione solution, dithiothreitol, and tris(2-carboxyethyl)phosphine are used as eluents to break the disulfide bonds on the surface of the magnetic microspheres, thereby removing the bonded target and retaining the polymer layer with a specific recognition cavity. The obtained immune agonist can target and recognize the siglec-10 receptor on the surface of macrophages and exhibit multiple functions: on the one hand, through the specific recognition cavity within the polymer matrix, the immune agonist can achieve efficient binding to macrophages in vivo; on the other hand, under the action of an external magnetic field, the immune agonist can rapidly aggregate at the tumor site, thereby anchoring the bound macrophages at the tumor site and prolonging the retention time, thus significantly enhancing the endogenous phagocytic ability of macrophages. In addition, macrophages that have taken up the immune agonist are effectively induced to polarize towards the M1 phenotype, further activating their anti-tumor immune function. The present invention has successfully developed an innovative therapeutic drug for TAMs, which not only significantly enhances the anti-tumor effect of macrophages but also minimizes the impact on normal tissues.

[0006] The preparation method of the multifunctional immune agonist for reprogramming tumor-associated macrophages is as follows: Prepare a mixed reaction solution with a concentration of 13 - 65 mM of N-isopropylacrylamide (NIPAm), functional monomer, N,N'-methylenebisacrylamide (Bis), and N-tert-butylacrylamide (TBAm), where the molar ratio of N-isopropylacrylamide, functional monomer, N,N'-methylenebisacrylamide, and N-tert-butylacrylamide is 32 - 98:1 - 45:1 - 5:1 - 20; then add a solid-phase carrier, and under the condition of continuously passing nitrogen, add 1 - 10 mM of ammonium persulfate (APS), and stir and polymerize the reaction at 38 - 45 °C for 8 - 16 h with an open mouth, and then remove the solution by magnetic separation. Finally, add a 10 - 20 mM thiol reducing agent solution to the nanoparticles and place them in an environment of 0 - 8 °C, and elute for 12 - 24 h to obtain the product.

[0007] The functional monomer is N-acryloyl-L-tyrosine (AcTyr) and / or N-acryloyl-L-phenylalanine (AcPhe).

[0008] The solid-phase carrier is a polypeptide linked to mercapto-functionalized magnetite nanoparticles.

[0009] The core amino acid sequence of the epitope polypeptide is CFRVERGSYVRYNFMND.

[0010] The preparation method of the solid-phase carrier is as follows: Prepare a polypeptide into a 0.01 - 0.5 mM solution with a 0.01 - 0.5 M NH 4 HCO 3 solution, then add mercapto-functionalized magnetite nanoparticles, stir and react at 20 - 30 °C for 4 - 8 h, and then remove the solution by magnetic separation. The above process is repeated at least 2 times.

[0011] The particle size of the mercapto-functionalized magnetite nanoparticles is 40 - 80 nm.

[0012] The stirring speed is 650 - 1200 rpm.

[0013] The thiol reducing agent is one or more of glutathione, dithiothreitol, and tris(2-carboxyethyl)phosphine. The present invention has the following beneficial effects:

[0014] (1) The present invention realizes the effective elution of the target through a cleavable disulfide bond, solves the problem of low recognition efficiency caused by difficult template elution in the prior art. The affinity K D value of the obtained immune agonist for the siglec-10 receptor can reach 10 -12 M, which is nearly 1000 times the binding affinity with a control protein (for example, human serum albumin). It can achieve efficient recognition of the siglec-10 receptor on the surface of macrophages.

[0015] (2) The multifunctional immune agonist obtained in the present invention can rapidly aggregate at the tumor site in response to an external magnetic field, achieving precise targeting of the tumor microenvironment. In vivo targeting experiments in animals have demonstrated that after being fluorescently labeled, this multifunctional immune agonist can successfully target macrophages at the tumor site in mice, achieving precise targeting of the tumor microenvironment. Subsequently, the macrophages bound by this multifunctional immune agonist are anchored at the tumor site and have an extended retention time, which promotes the intrinsic phagocytosis of macrophages.

[0016] (3) After being taken up by macrophages, the multifunctional immune agonist obtained in the present invention can effectively promote the polarization of macrophages towards the M1 phenotype, thereby activating the immunotherapeutic effect of macrophages. Therapeutic experiments in animals have shown that after treatment with this multifunctional immune agonist, the tumor growth inhibition rate exceeds 30%, and the survival rate of experimental mice exceeds 20% within 45 days. Description of the Drawings

[0017] Figure 1 1H NMR spectra of the synthesized functional monomers N - acryloyl - L - tyrosine (AcTyr) (top) and N - acryloyl - L - phenylalanine (AcPhe) (bottom).

[0018] Figure 2 Immune agonist SA 2 Flow cytometry results of binding to RAW264.7 cells (left) or BMDC cells (right).

[0019] Figure 3 Immune agonist SA 2 In vitro phagocytosis of tumor cells by macrophages RAW264.7 pretreated with (left) or monoclonal antibody mAb (right).

[0020] Figure 4 In vivo fluorescence imaging of mice.

[0021] Figure 5 Graphs of changes in tumor size (left), survival rate (middle), and body weight (right) during in vivo treatment of mice. Detailed Description of the Invention

[0022] 1. Preparation of the solid - phase carrier

[0023] Add 30 mg of thiolated magnetite nanoparticles (particle size 50 nm) to a 0.1 mM solution of the polypeptide (amino acid sequence CFRVERGSYVRYNFMND). The polypeptide solution is prepared with a 0.1 M NH 4 HCO 3 buffer solution. After stirring the reaction at 800 rpm at room temperature for 4 h, the solution is removed by magnetic separation; the above operation is repeated 3 times to ensure that the polypeptide is fully bound to the surface of the magnetic nanoparticles.

[0024] 2. Synthesis of the functional monomer N - acryloyl - L - tyrosine (AcTyr)

[0025] Dissolve L - tyrosine (10 mmol) in 8 mL of an aqueous NaOH (3.25 M) solution. Dropwise add acryloyl chloride (10 mmol) to the solution with constant stirring. Stir the mixed solution in ice water for 1 h, and then continue to stir at room temperature for 6 h. Extract the reaction mixture three times with 20 mL of ethyl acetate. Wash the organic phase twice with 20 mL of 0.1 M aqueous HCl solution, then dry, filter, and concentrate using a rotary evaporator to obtain a white, fluffy solid product. 1 H NMR (400 MHz, DMSO - d 6 ): δ 5.6, 6.0 (-CH 2 ), 6.25 (-CH), 8.3 (-NH), 4.4 (-CH), 12.6 (-COOH), 2.9 (-CH 2 ), 6.6, 7.0 -(C 6 H 4 ), 9.2 (-OH).

[0026] 3. Synthesis of the functional monomer N - acryloyl - L - phenylalanine (AcPhe)

[0027] Dissolve L - phenylalanine (0.020 mol) in 20 mL of 2 M aqueous sodium hydroxide solution and stir well. Subsequently, dropwise add acryloyl chloride (0.022 mol) to the mixed solution of L - phenylalanine and sodium hydroxide. During the addition of acryloyl chloride, keep the reaction mixture below 0 °C by external cooling with an ice bath. Then, continue to stir at room temperature for 2 h. Finally, filter, wash, and dry to obtain a white solid sample. 1 H NMR (400 MHz, DMSO - d 6 ): δ 5.6, 6.0 (-CH 2 ), 6.25 (-CH), 8.3 (-NH), 4.5 (-CH), 2.9, 3.1 (-CH 2 ), 6.6, 7.25 -(C 6 H 4 ).

[0028] Figure 1 The NMR results of the synthesized N - acryloyl - L - tyrosine (AcTyr) (upper figure) and N - acryloyl - L - phenylalanine (AcPhe) (lower figure) are shown. It can be proved from the figure that pure N - acryloyl - L - tyrosine (AcTyr) and N - acryloyl - L - phenylalanine (AcPhe) are successfully synthesized.

[0029] Example 1: Synthesis of Immunostimulant (SA 1 )

[0030] 1.02 mmol of NIPAm, 0.3 mmol of AcTyr (dissolved in 1 mL of aqueous NaOH (10 mM) solution), 0.03 mmol of Bis, and 0.075 mmol of TBAm were mixed with water to form a mixed reaction solution, and then 30 mg of solid support was added. The final volume of the reaction solution was 50 mL. Under continuous nitrogen flow, 0.26 mmol of APS was added, and the polymerization reaction was carried out with stirring at 40 °C for 12 h in an open system. After the reaction, the solution was removed by magnetic separation. After the reaction was completed, the supernatant was removed by magnetic separation. The obtained nanoparticle solution was transferred to a refrigerator at 4 °C, and an aqueous solution of glutathione at 10 mM was added, and the mixture was allowed to stand overnight. SA 1 was collected and reserved.

[0031] 2 mg of rhodamine B (dissolved in 1 mL of water) was added to the above pre-polymerized monomer solution, and other reaction conditions remained unchanged. SA labeled with rhodamine B was obtained. 1

[0032] Example 2: Synthesis of Immunostimulant (SA 2 )

[0033] 0.505 mmol of NIPAm, 0.065 mmol of AcPhe, 0.013 mmol of Bis, and 0.033 mmol of TBAm were mixed with water to form a mixed reaction solution, and then 30 mg of solid support was added. The final volume of the reaction solution was 50 mL. Under continuous nitrogen flow, 0.26 mmol of APS was added, and the polymerization reaction was carried out with stirring at 40 °C for 12 h in an open system. After the reaction, the solution was removed by magnetic separation. After the reaction was completed, the supernatant was removed by magnetic separation. The obtained nanoparticle solution was transferred to a refrigerator at 4 °C, and an aqueous solution of glutathione at 10 mM was added, and the mixture was allowed to stand overnight. SA 2 was collected and reserved.

[0034] 2 mg of rhodamine B (dissolved in 1 mL of water) was added to the above pre-polymerized monomer solution, and other reaction conditions remained unchanged. SA labeled with rhodamine B was obtained. 2

[0035] 4. Affinity of Immunostimulant for siglec-10 Protein Receptor

[0036] The K 1 and SA 2 values of the binding of immunostimulants SA D to siglec-10 protein were determined by bio-layer interferometry (BLI). As shown in Table 1, the affinities K 1 of SA 2 for siglec-10 protein D ​​The value can reach 10 -11 or more.

[0037] Table 1 BLI results of immune agonists binding to siglec-10 protein

[0038]

[0039] Immune agonist SA 1 and SA 2 Selectivity: Human serum albumin (HSA, pI = 4.6), γ-globulin (Glo, pI = 6.9), fibrinogen (Fib, pI = 5.5), myoglobin (Myo, pI = 7.0), ribonuclease A (RNA, pI = 8.9) were selected as control proteins, and the affinity K 1 values of SA 2 and SA D for these five proteins were tested by BLI. The results are shown in Table 2 and Table 3. The binding values of SA 2 to these proteins are between 10 -8 and 10 -9 which is about 1000 times different from the affinity of SA 2 with Siglec-10. In contrast, the binding values of SA 1 to these proteins are 10 -9 -10 -11 . These findings indicate that SA 2 has excellent binding affinity for siglec-10 promoted by electrostatic interaction, hydrogen bond interaction and hydrophobic interaction.

[0040] Table 2 BLI results of immune agonist SA 1 binding to control proteins

[0041]

[0042] Table 3 BLI results of immune agonist SA 2 binding to control proteins

[0043]

[0044] 5. In vitro cell experiments

[0045] 5.1 Binding effect of SA 2 and cells

[0046] Mouse macrophage RAW264.7 was selected as the positive expression cell of siglec-10 receptor, and mouse dendritic cell BMDC was selected as the negative expression cell of siglec-10 receptor. Flow cytometry experiments were performed using rhodamine B-labeled SA 2The dispersion (120 μg / mL) was used to stain RAW264.7 cells for 1 h, with untreated cells as the control. To ensure siglec-10 positive-expressing cells, the cells were also stained with an anti-siglece-10 antibody labeled with phycoerythrin (PE) for 1 h. Subsequently, the cells were washed, digested, and collected for flow cytometry analysis. From Figure 2 the flow cytometry results, it can be seen that the positive binding rate of SA 2 to RAW264.7 cells was 76.37%, close to the binding of a commercial protein antibody (mAb) to RAW264.7 cells, with a positive rate of 85.62%. While SA 2 barely bound to siglec-10 negative-expressing cells, BMDC cells, with a positive binding rate of only 3.14%.

[0047] 5.2 Experiment on the phagocytosis of tumor cells by macrophage RAW264.7 in vitro

[0048] Mouse ovarian cancer tumor cells ID8 were labeled with GFP. Macrophage RAW264.7 cells were labeled with pHRodoRed dye. The pHRodoRed dye is a pH-sensitive fluorescent dye, and the low pH of lysosomes causes the fluorescent dye to produce a red fluorescent signal when macrophages phagocytose tumor cells. After incubating macrophages with SA 2 (120 μg / mL) or sterile phosphate buffer (PBS, pH = 7.4) for 1 h, the cells were washed. After digesting the pretreated macrophages, they were co-cultured with tumor cells (10 5 cells / dish) at a ratio of 2:1 at 37 °C. After culturing for 12 h, 24 h, and 36 h, the cells were collected, washed, and analyzed by flow cytometry.

[0049] From Figure 3 the results, it can be seen that after co-incubating mouse ovarian cancer cells ID8 with SA 2 pretreated mouse macrophages RAW264.7 for 12 h, the macrophages had obvious phagocytic activity towards tumor cells, with a phagocytosis rate of about 50%. In contrast, macrophages treated with PBS had no obvious tumor phagocytosis. This indicates that SA 2 significantly enhanced the ability of macrophages to clear tumor cells by blocking the immune escape signal of tumors. In addition, as the co-culture time was extended to 24 h, the clearance rate of SA 2 pretreated macrophages towards tumor cells reached about 35%. When the co-culture time reached 36 h, the survival rate of tumor cells dropped sharply to below 20%. It is worth noting that by comparing with the phagocytosis effect of the monoclonal antibody (mAb) treatment group, we observed that the synthesized SA 2 achieved an effect equivalent to that of the antibody.

[0050] 6. Immune agonist SA 2 In vivo targeting tumor microenvironment experiment

[0051] 6.1 Synthesis of Cy7 fluorescently labeled poly(N - acryloyl - L - phenylalanine) polymer (SA 2 @Cy7)

[0052] Prepare a mixed reaction solution by adding 0.505 mmol NIPAm, 0.065 mmol AcPhe, 0.013 mmol Bis, and 0.033 mmol TBAm to water, then add 30 mg of solid support and 0.0065 mmol APM, and the final volume of the reaction solution is 50 mL. Under continuous nitrogen flow, add 0.26 mmol APS, and stir the polymerization reaction at 40 °C with an open mouth for 12 h, then remove the solution by magnetic separation. After the reaction, remove the supernatant by magnetic separation. Transfer the obtained nanoparticle solution to a 4 °C refrigerator, and add 10 mM glutathione aqueous solution, and let it stand overnight. Collect the amino - modified SA 2 for standby use.

[0053] Fluorescent dye Cy7 - NHS and amino - modified SA 2 are mixed at equimolar concentration in phosphate - buffered saline (PBS, pH = 7.4) and reacted overnight at room temperature. After the reaction, transfer the obtained Cy7 - labeled nanoparticle solution to a dialysis bag (molecular weight cut - off value: 1000 Da) and dialyze for 3 days. Collect the Cy7 - labeled SA 2 (SA 2 @Cy7) for standby use.

[0054] 6.2 Fluorescent imaging in mice

[0055] Use healthy 4 - week - old nude mice to establish an ID8 ovarian cancer syngeneic transplantation mouse model by subcutaneous injection of 10 7 cells into the right hind leg. Conduct the experiment when the tumor volume reaches approximately 100 mm 3 . The synthesized SA 2 @Cy7 is filtered through a sterile 0.2 - μm membrane with a concentration of 0.5 mg / mL. Use mice (n = 3 per group) to evaluate the in - vivo distribution of SA 2 @Cy7 over time. Inject approximately 200 μL of SA 2 @Cy7 via the tail vein, and apply an external magnetic field to the tumor site of the mice simultaneously. Perform fluorescent imaging on the mice at several time points after administration: 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h. Non - invasively monitor the whole - body fluorescent signal within four days to track the circulation and biodistribution kinetics of the nanoparticles.

[0056] From Figure 4 it can be seen that 12 h after intravenous injection, SA2 @Cy7 accumulates in the tumor microenvironment, indicating that SA 2 @Cy7 can successfully target macrophages in the tumor site in mice under the intervention of an external magnetic field, achieving precise localization of the tumor microenvironment.

[0057] 7. Immune agonist SA 2 In vivo tumor treatment experiment

[0058] A right tumor model was established using 4-week-old female BALB / c mice. First, 1×10 7 ID8 cells were subcutaneously injected into the right thigh of the mice to construct the tumor model. The mice were randomly divided into three groups of 5 each. When the tumor volume reached 50 mm 3 treatment was carried out. The treatment was performed every three days for a total of four times. The first group of mice was intravenously injected with PBS as the control group. The second group of mice was intravenously injected with an SA 2 dispersion (dose: 5 mg / kg). The third group of mice was intravenously injected with mAb (dose: 100 μg). The tumor volume, changes in mouse body weight, and survival rate were recorded.

[0059] From Figure 5 the results, it can be seen that compared with PBS, SA 2 can inhibit tumor growth and prolong the survival rate of mice, with a 20% survival rate at 45 days. This indicates that SA 2 has an effective effect on treating tumors. At the same time, by comparing the in vivo treatment effect of mAb, it can be seen that SA 2 is similar to mAb in terms of inhibiting tumor growth and prolonging the survival period of mice, which indicates that the tumor cell killing efficacy of synthetic SA 2 is completely comparable to that of commercial biological antibodies.

Claims

1. A method for preparing a multifunctional immune agonist for reprogramming tumor-associated macrophages, characterized in that: The specific operation of the preparation method is: N-isopropyl acrylamide, functional monomer, N,N'-methylenebisacrylamide, and N-tert-butyl acrylamide are prepared into a mixed reaction solution with a concentration of 13-65mM, wherein the molar ratio of N-isopropyl acrylamide, functional monomer, N,N'-methylenebisacrylamide, and N-tert-butyl acrylamide is 32-98:1-45:1-5:1-20; then a solid phase carrier is added, and under the condition of continuous nitrogen flow, 1-10mM ammonium persulfate is added, and the polymerization reaction is carried out at 38-45°C with open stirring for 8-16h, and then the solution is removed by magnetic separation; finally, a 10-20mM thiol reducing agent solution is added to the remaining nanoparticles, and the nanoparticles are placed in an environment of 0-8°C for elution for 12-24h to obtain the nanoparticles.

2. The preparation method according to claim 1, characterized in that: The functional monomer is N-acryloyl L-tyrosine and / or N-acryloyl L-phenylalanine.

3. The preparation method according to claim 1, characterized in that: The solid phase carrier is a polypeptide connected to thiolated ferrosoferric oxide magnetic spheres.

4. The preparation method according to claim 3, characterized in that: The core amino acid sequence of the polypeptide is CFRVERGSYVRYNFMND.

5. The preparation method according to claim 3, characterized in that: The preparation method of the solid phase carrier is as follows: the polypeptide is prepared into a 0.01-0.5 mM solution using a 0.01-0.5 M NH4HCO3 solution, and then thiolated ferrosoferric oxide magnetic balls are added, and the solution is removed by magnetic separation after stirring and reacting at 20-30° C. for 4-8 hours, and the above process is repeated at least twice.

6. The preparation method according to claim 5, characterized in that: The particle size of the mercaptolated ferroferric oxide magnetic spheres is 40-80 nm.

7. The preparation method according to claim 5, characterized in that: The stirring speed is 650-1200 rpm.

8. The preparation method according to claim 1, characterized in that: The thiol reducing agent is one or more of glutathione, dithiothreitol, and tris(2-carboxyethyl)phosphine.

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