CD47-targeting pharmaceutical composition as well as preparation method and application thereof

Abstract

The invention discloses a CD47-targeting pharmaceutical composition as well as a preparation method and application thereof, and belongs to the field of biological medicines. The pharmaceutical composition is HNP and comprises a polymer carrier, and a CD47 blocking agent and a PI3K gamma inhibitor which are wrapped in the polymer carrier. According to the CD47-targeting pharmaceutical composition, polypeptide (alphaCD47 peptide) is connected to the hydrophobic end of PC7A-AMA, after HNP is prepared, the polypeptide is wrapped inside, and immune-related adverse events caused by advanced falling of the polypeptide in the blood circulation process are reduced. The HNP improves the delivery of a hydrophobic drug IPI549, improves the accumulation of the drug at a tumor site, and enhances the curative effect; the alpha CD47 peptide can be released in response to a tumor microenvironment with a pH value of 6.5, and IPI549 can be released in response to a lysosome with a pH value of 5.3, so that efficient and accurate treatment of tumors can be realized. The HNP can promote the polarization of macrophages to M1 type, can greatly enhance the blocking effect of a CD47-SIRP alpha pathway, can effectively improve the phagocytosis and antigen presentation capability of the macrophages, can cause significant T cell infiltration and activation, can realize combined anti-tumor immunotherapy, and is high in safety.

Description

Technical Field

[0001] The present invention relates to a drug composition targeting CD47, a preparation method thereof and applications, and belongs to the field of biomedicine. Background Art

[0002] In recent years, innate immune checkpoints have become popular targets for cancer immunotherapy. Among them, the CD47-SIRPα signaling pathway, as an important mechanism for tumor cells to evade innate immune surveillance, has attracted much attention. CD47 is a transmembrane protein that is widely expressed on the surface of human cells and is overexpressed in a variety of tumor cells. Tumor cells use it to bind to a variety of proteins such as integrin, thrombospondin-1, and signal-regulatory protein alpha 1 (SIRPα) to exert immunosuppressive effects. Among these interactions, the interaction with SIRPα is particularly important. SIRPα is mainly expressed on the cell membranes of myeloid cells such as macrophages, dendritic cells, and monocytes. Overexpression of CD47 in tumor cells can bind to SIRPα on the surface of macrophages, generating a "Don't eat me" signal, thereby preventing phagocytosis by macrophages, which enables tumor cells to evade macrophage-mediated immune clearance. Aiming at this mechanism of action, developing CD47-targeted blockers to restore the phagocytic activity of macrophages is expected to achieve excellent cancer immunotherapy efficacy. However, in clinical trials, although CD47 blockers show good inhibitory effects on some hematological tumors, the therapeutic effects on solid tumors are still limited. This may be related to the widespread expression of CD47 on normal cells: for example, red blood cells also express CD47, and CD47 blockers may cause severe hematotoxicity at high doses. In addition, due to the long half-life of protein monoclonal antibodies and the inhibition of strong targets, the probability of adverse events in patients is increasing. To overcome the limitations of protein monoclonal antibodies, polypeptide antibodies have been gradually developed and applied due to their advantages of easy synthesis, loading, low immunogenicity, and strong tissue penetration. Nearly half of the contact surface between CD47 and SIRPα is located in 8 residues in the β-hairpin loop within the immunoglobulin domain of CD47. Replacing the central Thr with hydrophobic Phe to prepare a polypeptide with the sequence VTELFREG can effectively increase the affinity and block the CD47-SIRPα interaction. However, the current experimental data of immune checkpoint CD47-SIRPα inhibitors show that we still need to further search for more suitable and excellent candidate drugs.

[0003] In solid tumors, macrophages play important roles and can exhibit distinct functions through different polarization states. Tumor-associated macrophages (TAMs) are divided into M1 and M2 types. Among them, M1 type is also called classically activated macrophages, which can secrete a large amount of pro-inflammatory cytokines (such as IL-12, TNF-α). The secreted cytokines can not only directly kill tumor cells, but also activate other immune cells, such as CD8 + T cells and enhance their anti-tumor activities. In contrast, M2-TAMs are usually called alternatively activated macrophages, which secrete anti-inflammatory cytokines (such as IL-10) and growth factors (such as VEGF), thus promoting tumor growth, angiogenesis and immunosuppression. And M2-TAMs show upregulation of lysosomal function, which is prone to antigen degradation. In the tumor microenvironment (TME), tumor-promoting M2-TAMs are dominant, and the accumulation of M2-TAMs is often associated with poor prognosis, which reflects the importance of TAM polarization strategies. At the same time, M1-TAMs are more sensitive to CD47 blockers due to their naturally stronger phagocytic activity and lower lysosomal function. When the CD47 signal is blocked, M1-TAM can quickly recognize and phagocytose tumor cells, produce antigen-presenting peptides suitable for presentation to T cells, provide the initial antigen for cross-presentation to T cells to activate adaptive immunity, and further amplify the anti-tumor immune response by releasing pro-inflammatory factors. The polarized M1 macrophages are not only more easily activated by αCD47, but also can effectively eliminate tumor cells through their powerful phagocytic function, enhance antigen-presenting ability, and continuously activate subsequent adaptive immune responses. This result suggests that the combination of CD47-SIRPα inhibitors and TAM polarization regulators will further improve the efficacy of tumor immunotherapy.

[0004] The phosphatidylinositol-3-kinase (PI3K) / protein kinase B (AKT) signaling pathway is one of the important intracellular signal transduction pathways and is overactivated in various tumor types. PI3K is an important family of signal transduction enzymes, which are divided into three classes: class I, class II, and class III. Among them, class I PI3K is most relevant to cancer and is divided into class IA (including PI3Kα, β, and δ subtypes) and class IB (composed of the PI3Kγ subtype). The downstream signal transduction regulates cell proliferation, survival, migration, and metabolism through effector proteins (such as AKT and mammalian target of rapamycin (mTOR)), promoting the growth and spread of tumor cells. Inhibiting this pathway has become a hot topic in cancer prevention and cancer targeted therapy. In addition, PI3Kα and PI3Kβ are expressed in various cells including epithelial cells, PI3Kδ is expressed in T lymphocytes, and PI3Kγ is uniquely expressed in myeloid cells. In macrophages, PI3Kγ has been shown to play a crucial role in regulating cell activation and the formation of an inhibitory tumor microenvironment. By promoting the polarization of macrophages into the immunosuppressive M2 type, it inhibits the anti-tumor immune response. Inhibiting PI3Kγ can reprogram tumor-associated macrophages (TAMs) into the anti-tumor M1 type. IPI549 is a highly selective PI3Kγ inhibitor and has entered phase II clinical trials. However, IPI549 is extremely insoluble in water, only soluble in some organic solvents, has poor oral bioavailability, and has certain liver toxicity. There is an urgent need to make it fully exert its efficacy through appropriate delivery means.

[0005] Since the action site of immune checkpoint inhibitors (ICIs) is the outer membrane protein of tumor cells, and PI3Kγ inhibitors need to enter the cell to promote macrophage polarization, the different action sites make it difficult for the combination therapy strategy to achieve the expected effect. Therefore, it is necessary to formulate a multi-target combination therapy strategy through intelligent drug delivery design, enabling drugs with different action mechanisms to be released at specific sites, reducing the blood toxicity of systemic administration of αCD47 peptide, and at the same time improving the bioavailability of IPI549, which will better exert the advantages of combination therapy of the preparation. Summary of the Invention

[0006] Object of the Invention: The object of the present invention is to provide a pharmaceutical composition targeting the tumor-associated macrophage immune checkpoint CD47-SIRPα, its preparation method, and application. The pharmaceutical combination can regulate the polarization of macrophages from the M2 phenotype to the M1 phenotype, significantly enhance the blocking effect of the CD47-SIRPα pathway, effectively improve the phagocytosis and antigen presentation ability of macrophages, release tumor-killing cytokines, and trigger a highly efficient anti-tumor immune response.

[0007] Technical solution: The drug composition targeting CD47 of the present invention, the drug composition is HNP, including a polymer carrier, and a CD47 blocker and a PI3Kγ inhibitor encapsulated inside the polymer carrier.

[0008] Further, the polymer carrier includes PC7A-AMA and PDBA.

[0009] Further, the CD47 blocker is a polypeptide targeting the CD47-SIRPα signaling pathway.

[0010] Further, the amino acid sequence of the polypeptide is VTELFREG.

[0011] Further, the PI3Kγ inhibitor includes one or more of CZC24832, AS-604850 or IPI549.

[0012] The present invention also provides a preparation method of the above-mentioned drug composition targeting CD47, including the following steps:

[0013] (1) Dissolve formylbenzoic acid, EDC and NHS in DMF together, carry out carboxyl activation, then add PC7A-AMA, and add the polypeptide after the reaction ends to obtain PC7A-αCD47; the amino acid sequence of the polypeptide is VTELFREG;

[0014] (2) Mix PDBA, IPI549 and PC7A-αCD47 evenly, and slowly drop the mixture into pure water under probe sonication to form micelles. Ultrafilter the product, discard the lower layer liquid, add pure water again for ultrafiltration, and add pure water to the upper layer liquid to obtain the HNP liquid.

[0015] Further, the mass ratio of PC7A-AMA to the polypeptide is 10:1 to 15:1; the mass ratio of PC7A-AMA to PDBA in the drug composition is 2:1 to 1:2; IPI549 accounts for 6%-9% of the total mass in the drug composition, and the mass ratio of IPI549 to the polypeptide is 1:1 to 1:2.

[0016] Specifically, PC7A-AMA is prepared by the following steps: Dissolve C7A, AMA, PMDETA and MeO-PEG 114 -Br, 2-propanol and DMF, remove oxygen through three freeze-pump-thaw cycles, then add CuBr under a nitrogen atmosphere. After the polymerization reaction ends, dilute the reaction mixture with THF, remove the catalyst and THF solvent, dialyze the residue in distilled water and then freeze-dry to obtain a white powder, which is PC7A-AMA. The preparation method of PDBA is the same as that of PC7A-AMA, replace C7A with DBA and remove AMA.

[0017] The present invention also provides the use of the above-mentioned CD47-targeting pharmaceutical composition in the preparation of drugs for treating tumors.

[0018] Furthermore, the use is to regulate the polarization of tumor-associated macrophages from M2 to M1 type.

[0019] Furthermore, the content of IPI549 in the drug is 15 μg and the content of the polypeptide is 10 μg.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The CD47-targeting pharmaceutical composition of the present invention connects the polypeptide (αCD47 peptide) to the hydrophobic end of PC7A-AMA, and after preparing it into HNP, the polypeptide is wrapped inside, reducing the immune-related adverse events caused by the premature shedding of the polypeptide during blood circulation. HNP improves the delivery of the hydrophobic drug IPI549, increases its accumulation at the tumor site, and enhances the efficacy; and it can release the αCD47 peptide in response to the tumor microenvironment at pH ~ 6.5 and release IPI549 at lysosomal pH ~ 5.3, achieving efficient and precise treatment of tumors. HNP can promote the polarization of macrophages into M1 type, significantly enhance the blocking effect of the CD47-SIRPα pathway, effectively improve the phagocytosis and antigen presentation ability of macrophages, cause T cell infiltration and activation, achieve combined anti-tumor immunotherapy, and have high safety. Description of the Drawings

[0021] Figure 1 It is the 1H nuclear magnetic resonance spectrum of two polymer excipients;

[0022] Figure 2 It is the schematic diagram for the preparation of the nanoformulation HNP;

[0023] Figure 3 It is the particle size distribution of the dual-drug nanoparticles HNP in Example 1 of the present invention ( Figure 3 A), TEM image ( Figure 3 B) and stability ( Figure 3 C);

[0024] Figure 4 It is the cumulative release curve of αCD47 peptide and IPI549 in vitro under different pH conditions in Example 2 of the present invention; among them, Figure 4 A is the in vitro cumulative release curve of IPI549; Figure 4 B is the in vitro cumulative release curve of αCD47 peptide;

[0025] Figure 5 It is the cytotoxicity result graph of the obtained HNP on BMDM (A) and MC38 (B) cells in Example 3 of the present invention;

[0026] Figure 6Verification of the inhibitory effect of IPI549 on the protein levels of the PI3K-AKT signaling pathway and flow cytometry verification of promoting M1 polarization of macrophages in Example 4 of the present invention;

[0027] Figure 7 Confocal verification of αCD47 peptide promoting macrophage phagocytosis in Example 4 of the present invention;

[0028] Figure 8 Example 5: Investigation of the ability of HNP nanoparticles to promote antigen presentation of M2 TAMs;

[0029] Figure 9 Graph showing the changes in tumor growth curves of MC38 tumor-bearing mice and macrophage-depleted mice treated with HNP in Example 6 of the present invention;

[0030] Figure 10 Flow cytometry analysis results of the effects of HNP on the proportion of macrophages, M1 / M2 changes, and antigen presentation in tumors in Example 7 of the present invention

[0031] Figure 11 For CD8 in tumors in Example 7 of the present invention + T cell infiltration and CD8 + Graph of flow cytometry analysis results of the effect of T cell activation degree;

[0032] Figure 12 Graphs of in vivo, ex vivo, and pulmonary nodule statistics of lung metastases in 4T1 tumor-bearing mice treated with HNP in Example 8 of the present invention;

[0033] Figure 13 Graphs of blood routine and blood biochemical results of mice treated with intravenous administration of HNP in Experimental Example 9 of the present invention. Detailed implementation manners

[0034] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.

[0035] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified. The present invention will be further described in detail below with reference to specific embodiments and the accompanying drawings, and with reference to the data. It should be understood that this embodiment is only for illustrative purposes of the present invention and does not limit the scope of the present invention in any way. In the following embodiments, various processes and methods not described in detail are conventional methods well known in the art. The materials, reagents, devices, instruments, equipment, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0036] 2-(Azepan-1-yl)ethyl methacrylate (C7A) and 2-(dibutylamino)ethyl methacrylate (DBA) were purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.,

[0037] N,N,N',N”,N”-Pentamethyldiethylenetriamine (PMEDTA) was purchased from Tokyo Chemical Industry Co., Ltd. (Shanghai),

[0038] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0039] N-Hydroxy succinimide (NHS) was purchased from Shanghai Xianding Biotechnology Co., Ltd.

[0040] MeO-PEG 114 -Br and 2-(Methacryloyloxy)ethylammonium chloride (AMA) were purchased from Merck KGaA.

[0041] N,N’-Dimethylformamide (DMF, analytical grade) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.

[0042] Tetrahydrofuran (THF, analytical grade) was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0043] IPI549 (PI3Kγ inhibitor) was purchased from MedChemexpress Biotechnology Co., Ltd.

[0044] CD47 inhibitor αCD47 peptide (VTELFREG), 95%, was purchased from Nanjing JiePeptide Biotechnology Co., Ltd.

[0045] Example 1 Investigation of the particle size, TEM and stability of HNP nanomicelles

[0046] The synthesis steps of the polymer excipient were as follows: C7A (0.94 g, 4.47 mmol), AMA (46.2 mg, 0.28 mmol), PMDETA (14 μL, 0.067 mmol) and MeO-PEG were added into a polymerization tube 114-Br (0.29 g, 0.056 mmol). Then, a mixed solvent of 2-propanol (1.2 mL) and DMF (1.2 mL) was added to dissolve the monomer and initiator. After three freeze-pump-thaw cycles to remove oxygen, CuBr (5.8 mg, 0.04 mmol) was added to the polymerization tube under a nitrogen atmosphere, and then the polymerization tube was sealed in vacuo. After the polymerization reaction was completed, the reaction mixture was diluted with 10 mL of THF, and the catalyst was removed by passing through a neutral Al2O3 column. The THF solvent was removed by rotary evaporation. The residue was dialyzed against distilled water and then lyophilized to obtain a white powder, which was PC7A-AMA. The obtained product was subjected to 1H-nuclear magnetic resonance (NMR) detection to analyze its chemical structure. PDBA was prepared in the same manner. DBA (0.77 g, 3.2 mmol), PMDETA (8.4 μL, 0.04 mmol) and MeO-PEG 114 -Br (0.2 g, 0.04 mmol) were added to the polymerization tube, and the specific method was the same as that for PC7A-AMA. The 1H-NMR spectra of the prepared PC7A-AMA and PDBA are shown as Figure 1 follows.

[0047] The preparation method of the dual-drug nanoparticles was as follows: 1 mg of formylbenzoic acid was taken, 7.7 mg of EDC and 1.6 mg of NHS were added, and they were dissolved in 0.5 mL of DMF and stirred in an ice bath for 3 h for carboxyl activation. Then, 17 mg of PC7A-AMA polymer was added and reacted at 45 °C for 24 h. After the reaction was completed, excess formylbenzoic acid, EDC and NHS were removed by ultrafiltration. 2.2 mg of αCD47 peptide was added and the above product was continued to react at 45 °C for 24 h. Free polypeptides were removed by ultrafiltration to obtain the product PC7A-αCD47, which was dissolved in methanol for storage.

[0048] 5 mg of PDBA was taken, 160 μL (800 μg) of IPI549 and 0.5 mL (5 mg) of the PC7A-αCD47 mother liquor were added, and after mixing well, it was slowly dropped into 5 mL of pure water under probe sonication. The product was transferred to a 100 kDa ultrafiltration tube and ultrafiltered at 4000 rpm for 20 min. The lower layer liquid was discarded, and 3 mL of pure water was added to wash the micelles. After ultrafiltration, the lower layer liquid was discarded, and 2 mL of pure water was added to the upper layer to prepare the dual-drug nano-micelle HNP mother liquor (5 mg / mL). The preparation process of the nano-formulation HNP is shown as Figure 2 follows.

[0049] The HNP was resuspended with pure water, and the hydrated particle size and TEM morphology were characterized. The results are shown as Figure 3 follows: The particle size of the micelles was measured by dynamic light scattering method. After encapsulating IPI549 and αCD47 peptide, the hydrated particle size of the formed HNP was 40 nm.

[0050] Detection method of morphology: Pipette 30 μL of the sample and drop it on a 200-mesh copper grid. Use filter paper to absorb the excess sample on the copper grid. Drop 30 μL of phosphotungstic acid staining solution and stain for 2 min. Use filter paper to absorb the excess staining solution, dry it under an infrared drying lamp for 10 min, and place it under a transmission electron microscope Tecnai 12 for observation after natural drying. Figure 3 B is the transmission electron micrograph of HNP. TEM shows that the morphology of HNP is spherical, with a size of about 40 nm, small particle size and uniform dispersion.

[0051] Place HNP in PBS containing 10% FBS at 37 °C and measure the particle size of the micelles at different time points. Figure 3 C is the particle size change diagram of HNP. It can be seen from the figure that there is no significant change in the particle size of HNP, indicating that the micelle solution has good stability.

[0052] Investigation on the in vitro release characteristics of HNP in Example 2

[0053] Add the HNP micelles prepared in Example 1 into a dialysis bag (MW: 7000), place it in a 50 mL centrifuge tube, add 20 mL of PBS with pH 7.4, 6.5, and 5.0 respectively, and perform the release of αCD47 peptide in a shaker at 37 °C. At the set time points (5 min, 10 min, 20 min, 40 min, 1 h, 2 h, 3 h, 6 h, 9 h, 12 h, 23 h, 28 h, 31 h, 36 h, and 48 h), pipette 200 μL of the release solution respectively, and supplement 200 μL of fresh release medium to keep the volume constant. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the BCA content and draw the release curve. Add HNP into a dialysis bag (MW: 7000), place it in a 50 mL centrifuge tube, add 20 mL of PBS with pH 7.4, 6.5, and 5.0 respectively, and perform the release of IPI549 in a shaker at 37 °C. At the set time points (0.5 h, 0.75 h, 1 h, 2 h, 3 h, 5 h, 8 h, 12 h, 23 h, 26 h, 29 h, 35 h, 38 h, and 48 h), pipette 200 μL of the release solution respectively, and supplement 200 μL of the release medium. Use high-performance liquid chromatography (HPLC) to measure the content. The results are as Figure 4 shown. The in vitro release of the two drugs has pH responsiveness. The polypeptide is slowly released at pH 7.4, rapidly released at pH 6.5. At this time, IPI549 is still encapsulated in the micelles and is only rapidly released at pH 5.0, indicating that the drugs can be successfully released in vitro, the polypeptide can exert its efficacy under the conditions of the tumor microenvironment, and IPI549 enters macrophages to exert its efficacy.

[0054] Effect of HNP in Example 3 on the cell viability of BMDM and MC38 cells

[0055] (1) On the fifth day of culture, take BMDM (in 1640 medium containing 20 ng / mL mcsf) and MC38 cells (in 1640 medium) respectively, digest them with trypsin, count them under the microscope and inoculate them into 96-well plates, and culture for 24 h.

[0056] (2) Wash twice with PBS, add 100 μL of a series of pre-diluted and prepared free IPI549, IPI549@PDBA and HNP solutions (in terms of the concentration of IPI549, they are 0, 1, 2, 4, 8, 16, 32 μg / mL respectively) to each group, set 4 replicate wells, and set a zero-adjustment group without cells, and incubate for 24 h.

[0057] The IPI549@PDBA group: only loaded with IPI549, without αCD47 peptide, and the remaining preparation methods are the same as those of HNP.

[0058] (3) Add 10 μL of CCK8 solution to each well, gently shake the culture plate to mix evenly, and continue to incubate for 1 - 4 h

[0059] (4) Measure the absorbance value (OD) at a wavelength of 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Figure 5 The results of measuring cell viability by the CCK8 method are as follows. It can be seen from the figure that the cell viability of BMDM ( Figure 5 A) and MC38 ( Figure 5 B) is about 80% after administration of HNP, indicating that HNP has a weak inhibitory effect on cell growth and low toxicity.

[0060] Verification of the inhibition of the PI3K-AKT signaling pathway by IPI549 in Example 4, promotion of M2-M1 repolarization and promotion of macrophage phagocytosis by αCD47 peptide

[0061] 1. Detect the protein expression of PI3Kγ, AKT and P-AKT by Western blot experiment

[0062] M2-type BMDM cells were divided into a drug-free group and drug-containing groups (Free IPI549, IPI549@PDBA, HNP). The concentration of IPI549 in the drug-containing groups was 16 μg / mL. After drug administration and incubation for 24 h, the upper drug-containing culture medium of the cells treated with the drug was discarded. After rinsing three times with PBS, 100 μL of RIPA cell lysate was added to each well, and the cells were lysed at 4 °C for 30 min. During lysis, the cells were scraped off with a cell scraper. After 30 min, the lysate was collected in a 1.5 mL centrifuge tube and centrifuged at 4 °C and 12,000 rpm for 30 min, and then the supernatant was taken. The protein concentration of each sample was measured and calculated by the BCA method, and adjusted to a uniform concentration with RIPA lysate. The protein stock solution and 5× loading buffer were mixed at a volume ratio of 4:1 and heated in a 95 °C metal bath for 15 min to completely denature the protein. A precast protein electrophoresis gel was used, and the sample loading volume per well was 20 μL, and 5 μL of protein marker was loaded at both ends. The voltage was set at 70 V for 30 min and 120 V for 1 h.

[0063] The PVDF membrane was placed in methanol and activated for 30 s. After electrophoresis, they were stacked in the order of filter paper, PVDF membrane, gel, and filter paper in the transfer buffer, and the bubbles were removed. Finally, the power supply was turned on and set to the constant current mode, the current was set to 400 mA, and the transfer time was 25 min. The TBS buffer (10×) was diluted ten times and 0.1% (v / v) Tween 20 was added to prepare a TBST solution. The skimmed milk powder was diluted with TBST to a 5% blocking solution (w / v). The PVDF membrane was immersed in the blocking solution and blocked for 2 h. After blocking, according to the molecular weight indicated by the protein marker, the corresponding size of the band was cut and placed in the corresponding primary antibody solution (the dilution factors were rabbit-derived PI3 Kinase P110γ 1:1000, mouse-derived Phospho-AKT 1:5000, mouse-derived AKT 1:20000, mouse-derived GAPDH 1:5000), and incubated overnight at 4 °C. After the incubation of the primary antibody, it was washed with TBST, and the washing time was 10 min each time. After washing 4 times, the corresponding secondary antibody was added (the dilution factor of the secondary antibody was goat anti-rabbit 1:3000, goat anti-mouse 1:4000), and incubated at room temperature for 2 h. After the incubation of the secondary antibody, it was washed 4 times with TBST, 10 min each time. The ECL working solution A and B were mixed at a ratio of 1:1 to prepare a luminescent solution. After taking out the target band from TBST, the ECL luminescent solution was evenly dropped onto the band. A gel imaging system (TANON3900) was used to image the band. The results were as Figure 6 shown in A. IPI549 could effectively inhibit the PI3K signaling pathway and reduce AKT phosphorylation.

[0064] 2. Verification of macrophage repolarization

[0065] Extract BMDM and polarize them into M2, and administer drugs according to the set groups. Set up drug-free and drug-containing groups, prepare RPMI 1640 medium containing Free IPI549, IPI549@PDBA, and HNP with an IPI549 concentration of 16 μg / mL. In a biosafety cabinet, discard the original M2-induced medium in the 12-well plate, quickly add 1.5 mL of freshly prepared blank medium or drug-containing medium and label it, and place it in a 37°C, 5% CO2 cell culture incubator for continued culture for 24 h. Discard the cell culture supernatant, gently rinse twice with PBS, digest with trypsin, collect the cells, and resuspend the cells with a small amount of PBS, and transfer them to a 1.5 mL EP tube. Centrifuge the collected cells at 2500 rpm at 4°C for 5 min, and discard the supernatant.

[0066] Stain the M1 and M2 markers CD86 and CD206. Note that blank tubes without antibodies and single-stained tubes with only one specific antibody should be separated for adjusting voltage and compensation parameters, and incubate in the dark at 4°C for 30 min. After staining, centrifuge the cells at 500 g for 5 min and discard the supernatant. Finally, resuspend with 300 μL of PBS, and detect the fluorescence intensity of CD86- and CD206-positive by flow cytometry, and compare the polarization-promoting effects of different formulation groups. The results are as Figure 6 shown in Figures B and C. HNP can effectively deliver IPI549 combined with CD47 blockade to promote the polarization of M2 macrophages into M1 macrophages.

[0067] 3. αCD47 peptide-promoted macrophage phagocytosis experiment

[0068] For laser confocal microscopy, take MC38-Mcherry cells in the logarithmic growth phase, inoculate them in a 12-well plate at a density of 20×10 4 cells / mL, 1 mL per well. Polarized M2 BMDM are plated on chamber slides. After administering 1 mL of blank RPMI 1640 medium, Free αCD47, IPI549@PDBA, and HNP for 24 h, co-incubate in a 1:1 suspension of tumor:BMDM for 24 h, rinse 3 times with PBS, fix with paraformaldehyde for 15 min, then rinse 3 times with PBS. Add 250 μL of diluted CD11b-FITC antibody to each well for staining. After 30 min, rinse 3 times with PBS, stain with Hoechst for 15 min, wash 3 times with PBS, blot dry the residual moisture with filter paper, add a drop of mounting medium to each well and then cover with a cover slip, and observe it by laser confocal microscopy. The results are as Figure 7 shown. αCD47 peptide can restore the phagocytosis of macrophages on tumors. After using IPI549 to promote macrophage polarization, the phagocytosis can also be improved to a certain extent, and the combined administration group significantly promotes phagocytosis.

[0069] Example 5: Investigation of the ability of HNP nanoparticles to promote antigen presentation of M2 TAMs

[0070] Extract BMDM and polarize them into M1 and M2, and administer drugs according to the set groups. Using M1 TAMs as the positive control, set up drug-free and free IPI549, IPI549@PDBA, and IPI549@PDBA + αCD47 (equivalent to HNP, here to ensure that αCD47 can exert its efficacy extracellularly) dosing groups with an IPI549 concentration of 16 μg / mL. Place 1 mL of the culture volume in a cell culture incubator at 37 °C and 5% CO2 and continue culturing for 24 h. Then, in a biosafety cabinet, discard the drug-containing medium in the 12-well plate. After rinsing twice with PBS, add 300 μL of trypsin to each well. After terminating the digestion with complete medium, collect the cells and transfer them to 1.5 mL EP tubes. Centrifuge the collected cells at 500 g for 5 min at 4 °C, discard the supernatant, wash each tube once with 1 mL of PBS, and centrifuge at 500 g for 5 min at 4 °C to collect the cell pellet.

[0071] Stain with a mixture of CD11b-FITC, SIINFEKL-H-2kb-PE, and CD80-AF647 antibodies at 4 °C for 30 min. Note that blank tubes without antibodies and single-stained tubes with only one specific antibody need to be separated for adjusting voltage and compensation parameters. After staining, centrifuge the cells at 500 g for 5 min, discard the supernatant. Finally, resuspend with 300 μL of PBS and detect the fluorescence intensity of CD11b-positive H-2kb ( Figure 8 A) and CD80 ( Figure 8 B) by flow cytometry to compare the effect of the preparation on the antigen presentation ability of M2 macrophages. The dosing groups can increase the antigen presentation level of M2 macrophages and upregulate the macrophage activation level.

[0072] Example 6: Investigation of the anti-tumor ability of HNP nanoparticles

[0073] 1. Investigate the efficacy of HNP on a mouse colorectal cancer model

[0074] Inoculate 8 × 10 5 MC38 cells on the right side of the back of C57BL / 6 mice (6 - 8 weeks old, 18 - 22 g, Vital River) to establish an MC38 tumor-bearing mouse model. When the tumor grows to 60 - 80 mm 3 , randomly divide them into a normal saline group, free IPI549, free αCD47 peptide, IPI549@PDBA, αCD47@PC7A, and HNP groups, with 5 mice in each group. Take 5 mg of the PC7A-αCD47 reaction solution, and the specific preparation method is the same as that of HNP, which is αCD47@PC7A.

[0075] The day when the model was established was set as day 0. Administration was performed by intratumoral injection once every 3 days. The tumor volume was measured and recorded. Treatment was performed 4 times continuously. The tumor volume growth curve was plotted, and the body weight of the mice was monitored. Tumor volume calculation formula: V = length × width 2 / 2. Among them, the specification of the HNP injection is 50 μL per needle, and each needle contains 15 μg of IPI549 and 10 μg of αCD47 peptide. In the other groups, 15 μg of the drug IPI549 or 10 μg of αCD47 peptide is contained.

[0076] 2. Investigate the pharmacodynamic effect of HNP on the macrophage depletion model

[0077] After establishing the MC38 tumor-bearing mice, when the tumor grew to 60 - 80 mm 3 in size, they were randomly divided into a normal saline group, a macrophage depletion + HNP group, and an HNP group, with 5 mice in each group. For the construction of the macrophage depletion model, 200 μL of clodronate liposomes was injected via the tail vein every 6 days. After the macrophage depletion model was established, the day was set as day 0. Administration was performed by intratumoral injection once every 3 days. The tumor volume was measured and recorded. Treatment was performed 4 times continuously. The tumor volume growth curve was plotted, and the body weight of the mice was monitored. Tumor volume calculation formula: V = length × width 2 / 2. The specification of the HNP injection is 50 μL per needle, and each needle contains 15 μg of IPI549 and 10 μg of αCD47 peptide.

[0078] The pharmacodynamic results of the colorectal cancer model are as Figure 9 shown in A. After intratumoral administration, each nanoparticle group had an inhibitory effect on tumor growth. The HNP group had the best tumor inhibitory effect, and there were significant differences in tumor inhibitory effects compared with other groups. The pharmacodynamic effect of the macrophage depletion model is as Figure 9 shown in B. The HNP group had the best tumor inhibitory effect. In mice with macrophage depletion, treatment with HNP could not produce an effective tumor inhibitory effect, indicating that HNP exerts its anti-tumor efficacy through macrophages.

[0079] Example 7 Effects of HNP nanoparticles on the proportion of TAMs and antigen presentation at the tumor site, and on CD8 + T infiltration and activation

[0080] Investigate the effect of HNP on macrophages in tumor-bearing mice.

[0081] According to the method of colorectal cancer in Example 6, an MC38-OVA tumor-bearing mouse (C57BL / 6 mouse, 6 - 8 weeks old, 18 - 22 g, Vital River) model was established. When the tumor volume grew to 100 mm 3, randomly divided into saline group, free IPI549, IPI549@PDBA, αCD47@PC7A, and HNP groups, with 3 mice in each group. The day of model establishment was set as day 0, and different nanoparticles were administered on day 1 and day 4 respectively. Tumors were harvested on day 6, and the levels of TAM were analyzed by flow cytometry. Among them, the specification of the HNP injection was 50 μL per needle, and each needle contained 15 μg of IPI549 and 10 μg of αCD47 peptide. The other groups contained 15 μg of the drug IPI549 or 10 μg of αCD47 peptide.

[0082] The tumors were minced and placed in a digestive enzyme solution (1 mg / mL type IV collagenase, 0.3 mg / mL DNase) for 1 h of digestion. After terminating the digestion, the supernatant was discarded, and the precipitate was resuspended with 0.5% BSA / PBS, passed through a 70 μm cell strainer and ground to obtain a single-cell suspension. The density was adjusted to 1×10 7 cells / mL, and 0.5 mL of antibody mixture Zombie NIR Fixable Viability Kit, CD45-BV421, CD11b-BV605, F4 / 80-PercpCy5.5, CD206-APC, CD86-PE was added to each tube for TAM flow cytometry; Zombie NIR Fixable Viability Kit, CD45-BV421, CD11b-FITC, F4 / 80-PercpCy5.5, H2-KB-SIINFEKL-PE was used for macrophage antigen presentation flow cytometry; Zombie NIR Fixable Viability Kit, CD45-BV421, CD3-BV510, CD4-FITC, CD8-APC, and GZMB-PE were used for T cell flow cytometry. Stain in the dark on ice for 30 min, discard the supernatant, resuspend the cells, and perform on-machine detection. The results of TAM flow cytometry are as Figure 10 shown, where Figure 10 A is the proportion of macrophages in each group among immune cells, Figure 10 B is the proportion of M1 and M2 macrophages in each group, Figure 10 C is the proportion of H-2kb + TAM in each group.

[0083] The results showed that after treatment, there was no significant change in the proportion of CD11b + F4 / 80 + TAM among the groups. The proportion of CD86 + / CD206 + TAM in each group increased to varying degrees compared with the control group, and the M1 / M2 ratio in the HNP group was the highest, reaching 1.18. The MHCI-OVA + (i.e., H-2kb+ ) The proportion of TAM was significantly increased compared with the control group, reaching 22.7%, and the ability to promote antigen presentation by TAM was the most significant. The proportions of the other groups were 8.22% in the control group, 14.99% in the free IPI549 group, 18.4% in the IPI549@PDBA group, and 20.4% in the αCD47@PC7A group. The results showed that HNP had no obvious effect on the proportion of TAM in the tumor site, but could significantly promote the polarization of macrophages into the M1 type and enhance their antigen presentation level.

[0084] The flow cytometry results of T cells were as Figure 11 shown, among which Figure 11 A was the proportion of CD8 + T cells in immune cells in each group, Figure 11 B was the proportion of CD8 + / CD4 + T cells in each group, Figure 11 C was the proportion of GZMB + in CD8 + T cells in each group. After treatment, the proportion of CD8 + T cells in each group was significantly increased compared with the control group. Among them, the infiltration level of CD8 + T cells in the HNP group was the highest, reaching 6.1%. The proportion of GZMB + in CD8 + T cells in the HNP group was significantly higher than that in the control group, with a proportion of 25.1%, indicating that the activation degree of CD8 + T cells in the tumors of mice treated with HNP was the highest, meaning a stronger tumor killing effect.

[0085] Example 8 investigated the efficacy of HNP on different tumors

[0086] 5×10 5 4T1 cells were inoculated into the fat pad of the fourth pair of mammary glands of BALB / c mice to establish a mouse orthotopic breast cancer model. When the tumor volume grew to 60 - 80 mm 3 , the mice were randomly divided into a normal saline group, an IPI549@PDBA group, an αCD47@PC7A group, and an HNP group. Intratumoral injection was administered once every 3 days for 4 consecutive times. Among them, the specification of the HNP injection was 50 μL per needle, and each needle contained 15 μg of IPI549 and 10 μg of αCD47 peptide. The other groups contained 15 μg of the drug IPI549 or 10 μg of αCD47 peptide. The day of model establishment was set as day 0, and in vivo lung metastasis pictures were taken on days 21, 28, and 35 ([[]] Figure 12 A). On day 42, the mice were euthanized, the lungs were taken out, and ex vivo lung metastasis pictures were taken ([[]] Figure 12B), After fixation, observe the lung metastasis situation. Each drug administration group can inhibit the lung metastasis. The number of nodules in the HNP group ( Figure 12 C) was significantly less than that in the control group, proving that HNP can activate the systemic immunity and effectively inhibit the spontaneous lung metastasis of breast cancer.

[0087] Experimental Example 9 Safety Evaluation of Mice Treated with Intravenous Administration of HNP

[0088] Evaluate the in vivo safety of the nanoparticles based on the changes in blood biochemical indexes and blood routine. After intravenous tail vein injection of the mice in Example 8 four times, collect serum and whole blood. The results are as Figure 13 shown. There were no significant differences in the liver function indexes AST, ALT and the kidney function indexes CREA, UREA. The number of red blood cells and hemoglobin indexes in the HNP group were within the reference value range, while systemic administration of anti-CD47 monoclonal antibody could cause significant blood toxicity, and the number of red blood cells and hemoglobin was lower than the reference value range, indicating that HNP has good in vivo biosafety.

[0089] The present invention constructs a molecular mixed micelle HNP co-loaded with IPI549 and αCD47, which has high stability and biocompatibility, can be rapidly taken up by cells, and inhibits the PI3Kγ signaling pathway. It can effectively promote the polarization of macrophages to the M1 type at the in vivo tumor site, improve the immunosuppressive tumor microenvironment, and promote antigen presentation to activate CD8 + T cells. In the mouse colorectal cancer and triple-negative breast cancer models, HNP showed significant tumor inhibitory effects, effectively prolonging the survival period of mice. In addition, HNP can effectively avoid the blood toxicity caused by systemic administration of anti-CD47 monoclonal antibody drugs and has good safety. It inhibits the lung metastasis of breast cancer.

[0090] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as a limitation of the present invention itself. Without departing from the spirit and scope of the present invention defined by the appended claims, several modifications and refinements can be made in its form and details.

Claims

1. A drug composition targeting CD47, characterized in that, The pharmaceutical composition is HNP, which includes a polymer carrier, and a CD47 blocker and a PI3Kγ inhibitor encapsulated inside the polymer carrier.

2. The drug composition targeting CD47 according to claim 1, wherein The polymer carrier includes PC7A-AMA and PDBA.

3. The pharmaceutical composition targeting CD47 according to claim 1, characterized in that, The CD47 blocker is a polypeptide targeting the CD47-SIRPα signaling pathway.

4. The drug composition targeting CD47 according to claim 3, characterized in that, The amino acid sequence of the polypeptide is VTELFREG.

5. The drug composition targeting CD47 according to claim 1, characterized in that, The PI3Kγ inhibitor includes one or more of CZC24832, AS-604850, or IPI549.

6. A method for preparing the drug composition targeting CD47 according to any one of claims 1 to 5, characterized in that, It includes the following steps: (1) Dissolve formylbenzoic acid, EDC, and NHS in DMF together for carboxyl activation, then add PC7A-AMA, and after the reaction is completed, add the polypeptide to obtain PC7A-αCD47; the amino acid sequence of the polypeptide is VTELFREG; (2) Thoroughly mix PDBA, IPI549, and PC7A-αCD47 evenly, and slowly add the mixture dropwise to pure water under probe sonication to form micelles. Ultrafilter the product, discard the lower liquid, add pure water again for ultrafiltration, and add pure water to the upper liquid to obtain the HNP liquid.

7. The preparation method of the CD47-targeted pharmaceutical composition according to claim 6, characterized in that, The mass ratio of PC7A-AMA to the polypeptide is 10:1 to 15:1; the mass ratio of PC7A-AMA to PDBA in the pharmaceutical composition is 2:1 to 1:2; IPI549 accounts for 6%-9% of the total mass in the pharmaceutical composition, and the mass ratio of IPI549 to the polypeptide is 1:1 to 1:

2.

8. Use of the CD47-targeting pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a drug for treating tumors.

9. The application according to claim 8, characterized in that, The use is to regulate the polarization of tumor-associated macrophages from M2 to M1 type.

10. The application according to claim 8, characterized in that The content of IPI549 in the drug is 15 μg, and the content of the polypeptide is 10 μg.

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