A specific interfering polypeptide and its use

Abstract

The application discloses a specific interference polypeptide and application thereof. The amino acid sequence of the specific interference polypeptide is one of the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2: SEQ ID No. 1: NMEAQNTTEVYVK; and SEQ ID No. 2: QNTTEVYVK. The specific interference polypeptide provided by the application targets the αvβ3 / CD47-SIRPα interaction, has no side effect of lysing red blood cells and promoting tumor angiogenesis, and can be used for preparing a medicine for treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a specific interfering polypeptide and its applications. Background Technology

[0002] CD47 (integrin-associated protein, IAP) is a member of the immune supramolecular family and is a transmembrane glycoprotein located on the cell surface. It consists of an extracellular N-terminal immunoglobulin-variable IgV domain, a highly hydrophobic five-transmembrane segment, and a short C-terminal intracellular segment. CD47 is highly expressed in various malignant tumor cells, including myeloma, leiomyosarcoma, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, breast cancer, head and neck cancer, and osteosarcoma. CD47 overexpression is an independent predictor of poor prognosis in patients with various tumor types. CD47 expression is used by macrophages to distinguish between "self" and "non-self" tumors and interacts with signal regulatory protein α (SIRPα) on macrophages. The binding of CD47 to SIRPα promotes phosphorylation of tyrosine residues within the two typical immune receptor-based tyrosine-based inhibitory groups (ITIMs) encoded in the cytoplasmic tail of SIRPα. Subsequently, phosphorylated ITIMs recruit and activate protein tyrosine phosphatases (PTpases), particularly Src homology 2 (SH2) domain phosphatases SHP-1 and SHP-2. The interaction of the SH2 domain of these phosphatases with the phosphorylated ITIMs of SIRPα disrupts their autoinhibitory activity against the pTpase domain, thereby triggering enzyme activity. SIRPα also binds to SH2 domain-containing tyrosine phosphatases; both enzymes inhibit the accumulation of myosin IIA in phagocytic synapses and send a "don't eat me" signal to the innate immune system, thereby inhibiting macrophage-mediated phagocytosis and protecting normal cells from damage. This also represents a novel signaling mechanism used by tumor cells to evade detection and destruction.

[0003] In multicellular organisms, cell adhesion to the surrounding extracellular matrix (ECM) is crucial for tissue physiological development and functional integrity. Integrins are ubiquitous heterodimeric transmembrane glycoprotein adhesion receptors that play a multifaceted role as signaling molecules, mechanosensors, and key components of cell migration mechanisms, involved in virtually every step from primary to metastatic tumors. The ability of integrins to determine a cell's responsiveness to various inputs stems from their ability to differentially recognize different environments. To achieve this flexibility, each integrin consists of an α subunit and a β subunit, with 18 α subunits and 8 β subunits combined in stable, non-covalent linkages to generate 24 heterodimers known to have distinct functions. Based on different types of ECM components, integrins can be divided into two main categories: receptors that recognize Arg-Gly-Asp (RGD) peptide motifs and receptors that are independent of the RGD binding region. Eight integrins (αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, α8β1, and αIIbβ3) that recognize RGD motifs constitute the most important integrin receptor subfamily, participating in tumorigenesis and metastasis. Blocking integrin signaling has been shown to effectively inhibit tumor growth, angiogenesis, and metastasis. The conformation of integrins (inactive, activated, and a series of intermediate conformations), integrin-other related protein interactions, and processes such as endocytosis and recycling back to the plasma membrane can be altered by various factors, thus enabling them to participate more broadly in various physiological and disease processes. Among them, integrin αvβ3 is the most thoroughly studied integrin in its involvement in tumor progression, especially in tumor angiogenesis. Inhibiting αvβ3 signaling with antibodies (such as taracizumab, abituzumab, and intetumumab), peptides, or peptide mimics (such as cilengitide) and other small molecule antagonists shows promise in tumor therapy, antitumor agent delivery, or imaging of tumor lesions (using RGD peptide-based PET tracers). However, there are also side effects, such as low concentrations of αvβ3 antitumor agents potentially promoting tumor angiogenesis. While small molecules and peptides targeting integrins are currently used to treat fibrosis and ocular vascular diseases, antitumor agents targeting the αv series of integrins (especially αvβ3 and αvβ5) still have many safety and efficacy issues to be resolved.

[0004] αvβ3 and CD47 are highly expressed in various tumor types. While CD47 and αvβ3 have been considered as separate drug targets, their development in anti-tumor drugs has been challenging. αvβ3 actively regulates PD-L1 expression in the tumor microenvironment, thus playing a crucial role in immune evasion. αvβ3 deficiency inhibits the growth of primary tumors in mice, but currently, most tumor patients receiving anti-PD-1 or anti-PD-L1 therapy are unresponsive. Targeting αvβ3 may make tumors more sensitive to disruption of this axis, suggesting that besides PD-1 or PD-L1, there are many other unknown mechanisms by which αvβ3 participates in tumor immune escape. Red blood cells express CD47 but not αvβ3; in vitro protein immunoprecipitation has also demonstrated that αvβ3 and CD47 can directly interact. These preliminary results lead us to raise related questions, such as: Do the highly expressed αvβ3 and CD47 on the tumor cell membrane directly interact and form a functional association? Is this interaction static or dynamic? Is αvβ3 in an activated or inactivated state? This also leads to a bolder hypothesis: if αvβ3, which coexists with CD47, can regulate the function of the CD47-SIRPα immune checkpoint, then immune checkpoint therapy that specifically interferes with the CD47 / αvβ3 interaction on the surface of cancer cells would eliminate the hemolytic effect of CD47 antibodies acting on erythrocytes, as well as the effect of the SIRPα antibody Fc effect on myeloid cells (macrophages, monocytes), because it does not affect the function of erythrocytes (erythrocytes only express CD47). Furthermore, specifically interfering with the CD47 / αvβ3 interaction interface on the surface of cancer cells would also minimize the side effects of interfering with the binding of CD47 to other ligands, such as platelet-reactive protein-1 (TSP-1), vascular endothelial growth factor receptor 2 (VEGFR2), CD36, and Fas (CD95), or the effects of directly inhibiting αvβ3 on its normal function of recognizing ECM proteins containing the RGD tripeptide motif.

[0005] High expression of CD47 and ITGαvβ3 was found in various tumor cell lines (especially the triple-negative breast cancer cell line MDA-MB-231) and in 151 clinical samples of breast cancer and adjacent or normal tissues. Reducing the membrane expression of either CD47 or ITGαvβ3 on tumor cells significantly reduced the membrane distribution of the other, thereby weakening the CD47-SIRPα axis, increasing macrophage phagocytosis of cancer cells, and thus significantly inhibiting tumor growth.

[0006] CD47-SIRPα signaling enables malignant cells to evade macrophage-mediated phagocytosis; therefore, inhibiting the CD47-SIRPα signaling axis represents a promising strategy for cancer treatment. However, this treatment method, which has entered clinical trials, faces a series of challenges, including anemia, unclear upstream and downstream signaling mechanisms, varying effects on different tumors, and promotion of tumor angiogenesis. Summary of the Invention

[0007] Objective of the Invention: The objective of this invention is to provide a specific interfering peptide and its applications. The specific interfering peptide provided by this invention targets the αvβ3 / CD47-SIRPα interaction, without the side effects of erythrocyte lysis and tumor angiogenesis promotion, and can be used to prepare drugs for treating tumors.

[0008] CD47 / αvβ3 form a mutually stabilizing complex on the surface of cancer cells through direct interaction. This state can be simultaneously inhibited by antibodies against CD47, antibodies that activate αvβ3, the αvβ3 inhibitory peptides Cyclo(-RGDfk) and Cilengitide, and extracellular Ca2+. 2+ Interference with αvβ3 activation, such as knockdown of the key protein Talin, reduces CD47 and increases macrophage phagocytosis, suggesting that activated αvβ3 may interact with CD47. However, this state cannot be interfered with by the αvβ3 inhibitor RGDS. The extracellular ligand of αvβ3, fibronectin, can increase the distribution of the complex on the membrane and can also be affected by αvβ3 inactive antibodies. This indicates that αvβ3 interacting with CD47 is one or more special conformations independent of simple activated or inactive states.

[0009] This invention, through the study of the interaction mechanism of αvβ3 / CD47-SIRPα, discovered a mechanism that can target αvβ3 / CD47.

[0010] Specific interfering peptides of α-SIRPα interaction. These peptides can exert a therapeutic effect on tumors by reducing the expression of CD47 and αvβ3, thereby increasing the phagocytic activity of macrophages. Importantly, these compounds do not have the side effects of erythrocyte lysis or tumor angiogenesis promotion.

[0011] Technical solution: The objective of this invention is achieved through the following technical solution:

[0012] This invention provides a specific interfering polypeptide, wherein the amino acid sequence of the specific interfering polypeptide is the amino acid sequence shown in one of SEQ ID No. 1 to 2:

[0013] SEQ ID No. 1: NMEAQNTTEVYVK (abbreviated as: PSFL-NK13);

[0014] SEQ ID No. 2: QNTTEVYVK (abbreviated as: QK9).

[0015] The specific interference peptide of this invention is prepared according to existing technology.

[0016] The present invention also provides the application of the aforementioned specific interfering peptide in the preparation of drugs for treating tumors.

[0017] The present invention also provides the application of the aforementioned specific interfering peptide in combination with CD47 antibody in the preparation of drugs for treating tumors.

[0018] The tumor is breast cancer, small cell lung cancer, colorectal cancer, glioma, or leukemia.

[0019] The drug comprises a specific interfering polypeptide represented by one of the amino acid sequences SEQ ID No. 1 to 2, and a pharmaceutically acceptable carrier or excipient thereof.

[0020] The excipients include one or more of the following: emulsifiers, solubilizers, antioxidants, wetting agents, diluents, preservatives, disintegrants, binders, or lubricants.

[0021] The dosage form of the drug is tablets, granules, injections, capsules, oral liquids, or pills.

[0022] The medicament of the present invention can be administered in various known ways, such as orally, by injection, etc. The medicament of the present invention can be administered alone or in combination with other drugs. The oral composition can be any orally acceptable dosage form, including but not limited to tablets, granules, capsules, and oral liquids.

[0023] Sterile injectable compositions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Pharmaceutically acceptable carriers and solvents that can be used include water, sodium chloride solution, etc.

[0024] The actual dosage level of the active ingredient in the medicament of the present invention can be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and method of administration, and is non-toxic to the patient. The selected dosage level depends on a variety of factors, including route of administration, time of administration, excretion rate, duration of treatment, other drugs, compounds, and / or materials used in combination, the age, sex, weight, general health condition, and medical history of the patient being treated, as well as similar factors known in the medical field.

[0025] The specific interfering peptide, at 0.5-5 μM, reduces the expression of CD47 and αvβ3 and enhances the phagocytic activity of macrophages against tumor cells.

[0026] When the specific interfering peptide is used in combination with CD47 antibody at 0.5-5 μM, it enhances the effect of CD47 antibody in promoting macrophage phagocytosis of tumor cells.

[0027] The specific interfering peptide has no lytic effect on erythrocytes at 0.125-32 μM.

[0028] The specific interfering peptide does not promote tumor angiogenesis at 2nM-2μM.

[0029] The specific interfering peptide reduced tumor volume at a concentration of 0.5-8 mg / kg.

[0030] Beneficial effects:

[0031] The specific interfering peptides provided by this invention can significantly reduce the membrane expression of CD47 and αvβ3 in tumor cells, increase macrophage phagocytosis, slow the growth rate of orthotopic transplanted breast cancer in immunodeficient and normal mice, and are effective against various other tumors such as small cell lung cancer, colorectal cancer, and glioma. Most importantly, these peptides do not have the side effects of erythrocyte lysis or tumor angiogenesis promotion. Therefore, the specific interfering peptides of this invention can be used to prepare drugs for treating tumors. Furthermore, the effect of using the specific interfering peptides of this invention in combination with CD47 antibodies is stronger than using either one alone. Attached Figure Description

[0032] Figure 1 The expression of CD47 and αvβ3 was reduced by the specific peptide PSFL-NK-13; n = 3-4. * p<0.05 and *** p<0.001vs Ctrl.

[0033] Figure 2 The specific peptide PSFL-NK-13 increased macrophage phagocytosis of MDA-MB-231; n = 3-4, *** p<0.001vs Ctrl.

[0034] Figure 3 The combination of the specific peptide PSFL-NK-13 and CD47 antibody enhanced the phagocytic effect of CD47 antibody (B6H12) on tumor cells by macrophages. n=3, ***p<0.001 vs CD47 antibody (B6H12) group. Figure 4 To reduce CD47 in the specific peptide QK-9 ( Figure 4 A) and αvβ3 ( Figure 4 The expression of B) increased the phagocytosis of MDA-MB-231 by macrophages. Figure 4 C); n = 3-4, * p<0.05 vs Ctrl.

[0035] Figure 5 The specific peptide PSFL-NK-13 inhibits the expression of CD47 and αvβ3 in other tumor cells; Figure 5 AB flow cytometry was used to detect the expression of αvβ3 and CD47 on the HCT-116 cell membrane by PSFL-NK-13 at a concentration of 4 μM; Figure 5 CD flow cytometry was used to detect the expression of αvβ3 and CD47 on the A549 cell membrane by PSFL-NK-13 at a concentration of 4 μM; Figure 5 EF flow cytometry was used to detect the expression of αvβ3 and CD47 on the HL-60 cell membrane by PSFL-NK-13 at a concentration of 4 μM. n = 3-4. ** p<0.01vsCtrl.

[0036] Figure 6 The peptide PSFL-NK-13 inhibits the development of in situ breast cancer tumors in nude mice; Figure 6 A is a statistical graph of tumor volume-time curves for the Ctrl group and the three dosage groups of PSFL-NK-13 (0.5, 2 and 8 mg / kg); Figure 6 B is a schematic diagram of tumor size in the Ctrl group and the three dosage groups of PSFL-NK-13 (0.5, 2 and 8 mg / kg); Figure 6 C is a statistical graph of tumor weight in the Ctrl group and the three dosage groups of PSFL-NK-13 (0.5, 2 and 8 mg / kg); n=6. * p<0.05, ** p<0.01 and *** p<0.001vs Ctrl.

[0037] Figure 7 The peptide PSFL-NK-13 inhibited the progression of colorectal cancer tumors in nude mice; Figure 7 A is a statistical graph of tumor volume-time curves for the Ctrl group and the PSFL-NK-13 (2mg / ml) group; Figure 7 B is a schematic diagram of tumor size in the Ctrl group and the PSFL-NK-13 (2mg / kg) group; Figure 7 C is a statistical graph of tumor weight in the Ctrl group and the PSFL-NK-13 (2mg / kg) group; n=6. *** p<0.001vsCtrl.

[0038] Figure 8 This study compares the effects of peptides PSFL-NK-13 and B6H12 antibodies on the hemolytic effect of RBCs.

[0039] Figure 9The effects of peptides PSFL-NK-13 and Cilengitide on HUVEC tubule formation and statistical graphs. Detailed Implementation

[0040] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0041] Unless otherwise specified, all reagents or instruments used in this invention are conventional products that can be purchased on the market.

[0042] Example 1: Preparation of the specific interfering peptide PSFL-NK-13

[0043] The amino acid sequence of PSFL-NK-13 is: NMEAQNTTEVYVK.

[0044] Peptides were synthesized using solid-phase peptide synthesis technology: 5 g of Fmoc-aa-Wang resin (Jier Biochemical) was weighed and swollen in 10 times its weight of dichloromethane for 20 minutes. Then, 1.58 ml of deprotection solution (morphorline: N,N-dimethylformamide = 6:4, v / v) was added, and the reaction proceeded for 30 minutes. After the reaction, the resin was washed three times alternately with dichloromethane and N,N-dimethylformamide. Fmoc-amino acids (8.5 mmol, Jizhi Biochemical) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 10.2 mmol, Aladdin) were dissolved in 10.2 mmol of N,N-dimethylformamide, mixed thoroughly, and the reaction solution was added to the resin and reacted for 4 hours. After the reaction, the resin was washed three times alternately with dichloromethane and N,N-dimethylformamide. The mixture was then dried under a nitrogen stream. After drying, 200 mL of cleavage buffer (trifluoroacetic acid-deionized water-triisopropylsilane = 95.0:2.5:2.5, v / v) was added to the resin and the mixture was shaken for 1.5 h to cleave the target peptide from the resin. The target peptide was precipitated with ice-cold ether, and the crude peptide was collected by centrifugation and purified by HPLC.

[0045] The purity of the purified peptides was identified by high performance liquid chromatography (HPLC), the molecular weight was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and the amino acid sequence structure was determined by an automated amino acid sequencer.

[0046] PSFL-NK-13 peptide purity is above 95%;

[0047] [M+2H] 2+ : 764.20.

[0048] Example 2: Preparation of specific interfering peptide QK-9

[0049] The amino acid sequence of QK-9 is: QNTTEVYVK.

[0050] Polypeptide synthesis using solid-phase peptide synthesis: 5g of Fmoc-aa-Wang resin (Jier Biochemical) was weighed and swollen for 20 minutes with 10 times its weight of dichloromethane. Then, 1.58ml of deprotection solution (morphorline: N,N-dimethylformamide = 6:4, v / v) was added, and the reaction proceeded for 30 minutes. After the reaction, the resin was washed three times alternately with dichloromethane and N,N-dimethylformamide. Fmoc-amino acids (8.5mmol, Jizhi Biochemical) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 10.2mmol, Aladdin) were dissolved in 10.2mmol of N,N-dimethylformamide, mixed thoroughly, and the reaction solution was added to the resin and reacted for 4 hours. After the reaction, the resin was washed three times alternately with dichloromethane and N,N-dimethylformamide. The resin was then dried under a nitrogen stream. After drying, 200 mL of cleavage buffer (trifluoroacetic acid-deionized water-triisopropylsilane = 95.0:2.5:2.5, v / v) was added to the resin and the mixture was shaken for 1.5 h to cleave the target peptide from the resin. The target peptide was precipitated with ice-cold ether, and the crude peptide was collected by centrifugation and purified by HPLC.

[0051] QK-9 polypeptide purity is over 95%;

[0052] [M+2H] 2+ : 541.40.

[0053] Example 3: The specific interfering peptide PSFL-NK-13 reduces the expression of CD47 and αvβ3 on breast cancer cells MDA-MB-231 and enhances the phagocytic activity of macrophages on them.

[0054] 1. Flow cytometry was used to detect the expression of CD47 and αvβ3 on the surface of MDA-MB-231 cells.

[0055] Experimental methods:

[0056] MDA-MB-231 cells (purchased from ATCC) were seeded and cultured overnight at 37°C until good cell adhesion was achieved. Cells were then incubated with 4 μM PSFL-NK-13 or blank solvent (0.5% DMSO) for 48 h, followed by transfection with siRNA (Shanghai GenePharmaCo Ltd.). After medium change, cells were cultured for another 48 h. The supernatant was aspirated, and the cells were digested with trypsin (Gibco) and resuspended. The resuspended cells were then transferred to a 5 ml flow cytometry tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were washed once with 1 ml PBS and centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was carefully aspirated, and the cells were resuspended in 100 μl of the solution. In PBS, remove the flow cytometry antibodies FITC-CD51 / 61 (304402, Biolegend) and APC-CD47 (17-0479-42, Invitrogen) and place them on an ice box. Incubate at room temperature in a shaker for 30 min to fully label the cells with the antibodies. The shaker speed is 150 rpm. Resuspend the cells in 500 μl of PBS and place them on ice in the dark. Collect the cells using a flow cytometer (BD LSR-Fortessa). The data are analyzed using FlowJo 10.6 software.

[0057] Analysis of flow cytometry experimental results: such as Figure 1 As shown, compared with the control group Ctrl(DMSO), 4 μM peptide PSFL-NK-13 (cell treatment time 48 h) reduced the expression of integrin αvβ3 and CD47 on the MDA-MB-231 cell membrane in a dose-dependent manner. At a concentration of 4 μM, the expression of αvβ3 membrane protein decreased from 81.0±1.7% to 54.4±2.3%, and the expression of CD47 membrane protein decreased from 92.4±1.5% to 71.0±2.6%, showing significant differences between the two groups.

[0058] 2. Flow cytometry was used to determine the phagocytosis of tumor cells by macrophages.

[0059] Experimental methods:

[0060] THP-1 cells (derived from ATCC) were seeded in 24-well plates and phorbol 12-myristate 13-acetate (PMA, derived from MCE) was added to a final concentration of 200 ng / ml to induce them into macrophages. MDA-MB-231 cells (derived from ATCC) were treated with blank solvent (0.5% DMSO) or 4 μM PSFL-NK-13 for 48 h, followed by trypsin digestion, cell counting, and adjustment of the cell density to 2 × 10⁶ cells / well. 5Cells were counted at 500 μl / ml, and 500 μl of cell suspension was transferred to an EP tube. The cells were centrifuged at 1000 rpm for 5 min, and then resuspended in 500 μl of RPMI-1640 basal medium (C22400500BT, Gibco). The fluorescent probe Protonex was then added. TM Red 600 (21207, AAT Bioquest) was used to prepare a 5mM stock solution, which was added to the cell suspension to a final concentration of 0.5μM. The cells were incubated at 37°C for 30 min. RPMI-1640 was added to an EP tube, and the cells were centrifuged at 1000 rpm for 5 min. The cells were then resuspended in RPMI-1640 for later use. The supernatant of THP-1 cells was removed, and the above-mentioned MDA-MB-231 cell suspension was added. The cells were incubated together for 1 h. The cell supernatant was removed, and the cells were digested with trypsin, centrifuged at 1000 rpm for 5 min, washed once with PBS, and resuspended in 500 μl of PBS. Flow cytometry was used to detect the phagocytic activity of macrophages (THP-1) on tumor cells (MDA-MB-231).

[0061] Flow cytometry analysis results showed that: Figure 2 As shown, compared with the control group (DMSO), the proportion of THP1 cells phagocytosed by tumor cells significantly increased after 48 h of treatment with 4 μM peptide PSFL-NK-13, with a statistically significant difference between the two groups. The phagocytosis rate in the PSFL-NK-13 treatment group increased from 9.1±1.2% to 31.5±4.3%.

[0062] Example 4: The combination of the specific interfering peptide PSFL-NK-13 and CD47 antibody can enhance the effect of CD47 antibody in promoting macrophage phagocytosis of MDA-MB-231 cells.

[0063] THP-1 cells (derived from ATCC) were seeded in 24-well plates and phorbol 12-myristate 13-acetate (PMA, derived from MCE) was added to a final concentration of 200 ng / ml to induce them into macrophages. Separately, MDA-MB-231 cells (derived from ATCC) were treated with blank solvent (0.5% DMSO), 4 μM PSFL-NK-13, 10 μg / ml CD47 antibody (B6H12, Abcam), and 4 μM PSFL-NK-13 + 10 μg / ml CD47 antibody for 48 h, respectively. Cells were then digested with trypsin, counted, and adjusted to a cell density of 2 × 10⁶ cells / well. 5Cells were counted at 500 μl / ml, and 500 μl of cell suspension was transferred to an EP tube. The cells were centrifuged at 1000 rpm for 5 min, and then resuspended in 500 μl of RPMI-1640 basal medium (C22400500BT, Gibco). The fluorescent probe Protonex was then added. TM Red 600 (21207, AAT Bioquest) was used to prepare a 5mM stock solution, which was added to the cell suspension to a final concentration of 0.5μM. The cells were incubated at 37°C for 30 min. RPMI-1640 was added to an EP tube, and the cells were centrifuged at 1000 rpm for 5 min. The cells were then resuspended in RPMI-1640 for later use. The supernatant from THP-1 cells was removed, and the above-mentioned MDA-MB-231 cell suspension was added. The cells were incubated together for 1 h. The supernatant was removed, and the cells were digested with trypsin, centrifuged at 1000 rpm for 5 min, washed once with PBS, and resuspended in 500 μl of PBS. Flow cytometry was used to detect the phagocytic activity of macrophages (THP-1) on tumor cells (MDA-MB-231).

[0064] Flow cytometry analysis results showed that: Figure 3 As shown, compared with CD47 antibody (B6H12) alone, the combined treatment of 4 μM peptide PSFL-NK-13 with CD47 antibody (B6H12) for 48 h significantly increased the proportion of THP1 cells phagocytosed by tumor cells, with a statistically significant difference between the two groups. The phagocytosis rate increased from 27.1 ± 2.4% in the CD47 antibody (B6H12) alone group to 48.7 ± 4.4% in the combined treatment group.

[0065] Example 5: The specific interfering peptide QK-9 reduces the expression of CD47 and αvβ3 on breast cancer cells MDA-MB-231 and enhances the phagocytic activity of macrophages on them.

[0066] 1. Flow cytometry was used to detect the expression of CD47 and αvβ3 on the surface of MDA-MB-231 cells.

[0067] Experimental methods:

[0068] MDA-MB-231 cells were seeded and cultured overnight at 37°C until good cell adhesion was achieved. Cells were transfected with siRNA, and after medium change, they were cultured for another 48 hours. The supernatant was aspirated, and the cells were resuspended after trypsin digestion. The resuspended cells were then transferred to a 5 ml flow cytometry tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were washed once with 1 ml PBS and centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was carefully aspirated, and the cells were resuspended in 100 μl PBS. The flow cytometry antibodies FITC-CD51 / 61 and APC-CD47 were placed on ice and incubated on a shaker at room temperature for 30 min to ensure adequate antibody labeling. The shaker speed was 150 rpm. The cells were resuspended in 500 μl PBS and kept on ice in the dark. Cells were collected using a flow cytometer (BD LSR-Fortessa), and the data were analyzed using FlowJo 10.6 software.

[0069] 2. Flow cytometry was used to determine the phagocytosis of tumor cells by macrophages.

[0070] Experimental methods:

[0071] THP-1 cells were seeded in 24-well plates and inducing to morphology into macrophages by adding PMA to a final concentration of 200 ng / ml. After treatment with blank solvent (0.5% DMSO) or 4 μM QK-9 for 48 h, cells were digested with trypsin, counted, and the cell density was adjusted to 2 × 10⁶ cells / well. 5 Cells were counted at 1000 μl / ml, and 500 μl of cell suspension was transferred to an EP tube. The cells were centrifuged at 1000 rpm for 5 min, and then resuspended in 500 μl of RPMI-1640 basal medium. The fluorescent probe Protonex was added. TM Red 600 (AAT Bioquest) was prepared at a concentration of 5 mM and added to the cell suspension to a final concentration of 0.5 μM. The cells were incubated for 30 min. RPMI-1640 was added to an EP tube, and the cells were centrifuged at 1000 rpm for 5 min. The cells were then resuspended in RPMI-1640. The supernatant was removed, and MDA-MB-231 cell suspension was added for co-incubation. The cell supernatant was removed, and the cells were digested with trypsin and centrifuged at 1000 rpm for 5 min. The cells were washed once with PBS and resuspended in 500 μl of PBS. Flow cytometry was used to detect the phagocytic activity of macrophages on tumor cells.

[0072] Flow cytometry results: such as Figure 4As shown, compared with the control group Ctrl(DMSO), treatment with 4 μM peptide QK-9 for 48 h reduced the expression of integrin αvβ3 and CD47 on the MDA-MB-231 cell membrane. The proportion of tumor cells phagocytosed after QK-9 treatment significantly increased, showing a statistically significant difference between the two groups. The expression of integrin αvβ3 on the MDA-MB-231 cell membrane decreased from 80.7±3.0% to 70.1±4.8%, and the expression of CD47 decreased from 89.3±2.5% to 82.6±1.3%. The proportion of tumor cells phagocytosed after QK-9 treatment increased from 9.1±2.9% to 17.4±1.5%.

[0073] Example 6: Inhibition of CD47 and αvβ3 expression on rectal cancer HCT-116 cells, lung cancer A549 cells, and leukemia HL-60 cells by the specific interfering peptide PSFL-NK-13.

[0074] The expression of CD47 and αvβ3 on the surface of rectal cancer HCT-116 cells, lung cancer A549 cells, and leukemia HL-60 cells was detected by flow cytometry.

[0075] Rectal cancer HCT-116 cells, lung cancer A549 cells, or leukemia HL-60 cells (all from ATCC) were inoculated and cultured overnight at 37°C until good cell adhesion was achieved. Cells were then incubated with blank solvent (0.5% DMSO) or 4 μM SFL-NK-13 for 48 h. The supernatant was discarded, and the cells were resuspended after trypsin digestion. The resuspended cells were transferred to a 5 ml flow cytometry tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were washed once with 1 ml PBS and centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was carefully discarded, and the cells were resuspended in 100 μl PBS. Flow cytometry antibodies FITC-CD51 / 61 and APC-CD47 were placed on ice and incubated at room temperature on a shaker for 30 min at 150 rpm to ensure complete antibody labeling. The cells were then resuspended in 500 μl PBS and kept on ice in the dark. Flow cytometry was performed using a BD (Blood Flow Cytometer). Cells were collected using LSR-Fortessa, and the data were analyzed using FlowJo 10.6 software.

[0076] We first examined the inhibitory effect of the peptide PSFL-NK-13 on colorectal cancer cells HCT-116 and non-small cell lung cancer cells A549. For example... Figure 5As shown, at a concentration of 4 μM, the peptide PSFL-NK-13 can reduce the expression levels of integrin αvβ3 and CD47 on tumor cells. Specifically, in HCT-116 cells, αvβ3 expression decreased from 57.3±3.5% to 46.2±1.9%, and CD47 expression decreased from 61.7±1.9% to 50.1±2.5%. In A549 cells, αvβ3 expression decreased from 69.5±3.0% to 55.0±2.1%, and CD47 expression decreased from 76.2±2.0% to 63.6±2.7%.

[0077] In myeloid leukemia cells HL-60, αvβ3 expression decreased from 66.0±1.9% to 52.6±4.8%, and CD47 expression decreased from 78.0±3.3% to 67.6±1.9%. The peptide PSFL-NK-13 also reduced the expression of αvβ3 and CD47 in hematological malignancies (see...). Figure 5 AC).

[0078] Example 7: Specific interfering peptide PSFL-NK-13 inhibits the development of mammary carcinoma in situ in nude mice.

[0079] Test method:

[0080] A subcutaneous xenograft model was established in nude mice (8 weeks old, Jiangsu Huachuang Xinno) using orthotopic injection of MDA-MB-231 cells. MDA-MB-231 cells (derived from ATCC) were injected at a rate of 4 × 10⁶ cells / year. 6 The tumor cells were inoculated into the mammary fat pads of mice at a specified number per mouse. Two weeks after orthotopic transplantation, mice were randomly assigned to four groups based on tumor growth: a control group (Ctrl, saline), a low-dose PSFL-NK-13 group (0.5 mg / kg), a medium-dose PSFL-NK-13 group (2 mg / kg), and a high-dose PSFL-NK-13 group (8 mg / kg). Mice were initially treated with PSFL-NK-13 at doses of 0.5 mg / kg, 2 mg / kg, and 8 mg / kg, administered subcutaneously three times weekly for four weeks.

[0081] Four weeks after treatment, the tumor volume in all three dose groups was significantly reduced. Ex vivo tumor images were taken (see...). Figure 6 B) A significant reduction in tumor volume was observed in the treatment group, and compared with the control group (Ctrl), the tumor weight in the PSFL-NK-13 peptide treatment group was significantly reduced (see [link]). Figure 6 AC).

[0082] Example 8: Specific interfering peptide PSFL-NK-13 inhibits the development of colorectal cancer tumors in nude mice.

[0083] Test method:

[0084] A subcutaneous xenograft model in nude mice was established by subcutaneous injection of HCT-116 cells. HCT-116 cells (derived from ATCC) were injected at a rate of 1×10⁻⁶. 6 Tumor cells were inoculated subcutaneously in the abdomen of 8-week-old nude mice (Jiangsu Huachuang Xinno). One week after subcutaneous transplantation, the mice were randomly and uniformly divided into two groups based on tumor growth: a control group (Ctrl, saline) and a PSFL-NK-13 group (2 mg / kg). The PSFL-NK-13 peptide was administered to the nude mice at a dose of 2 mg / kg via daily local subcutaneous administration.

[0085] After three weeks of treatment, the tumor volume of nude mice treated with the peptide PSFL-NK-13 continuously decreased. Compared with the untreated group, the tumors treated with the peptide were significantly smaller, and the tumor weight showed a significant difference. The tumor size after dissection was illustrated in photographs. The tumor weight in the Ctrl group was 0.61±0.11g, and the tumor weight in the PSFL-NK-13 (2mg / kg) group was 0.24±0.12g. The difference in tumor weight between the two groups was significant (see...). Figure 7 AC).

[0086] Example 9: No hemolysis of red blood cells was observed with the specific interfering peptide PSFL-NK-13.

[0087] Blood was collected from healthy volunteers and processed using sampling tubes containing anticoagulant. The blood samples were then mixed with human lymphocyte separation medium (Tianjin Haoyang Biotechnology Co., Ltd., LTS10771). The bottom layer of blood cells was separated and collected, resuspended in PBS, centrifuged at 400g for 10 min, and washed twice. Red blood cells were mixed with PBS to prepare a 2.5% (v / v) cell suspension. 50 μl of this red blood cell suspension was added to a 96-well circular plate and mixed with 50 μl of either PSFL-NK-13 peptide at a concentration of 0.125–32 μM or CD47 antibody (B6H12) at a concentration of 0.3125–80 μg / ml. The plates were incubated at 37°C for 4–6 h. Hemagglutination was observed in the supernatant, with red flocculent material forming blood cell aggregation. No significant change was observed in the colorless supernatant or in the complete sedimentation of red blood cells. The middle field of view of the plate was photographed under a 100× white light microscope.

[0088] The red blood cell hemolysis experiment revealed: For example Figure 8 As shown, CD47 antibody (B6H12) at a concentration of 5 mg / ml can cause erythrocyte aggregation, hemolysis, and erythrocyte lysis. However, the corresponding dosage of peptide PSFL-NK-13 has no effect on erythrocytes and no hemolysis is observed. Even when the dosage is increased to 100 μM and 200 μM, no damage to erythrocytes is observed.

[0089] Example 10: The specific interfering peptide PSFL-NK-13 does not promote angiogenesis.

[0090] 10 mg / ml Matrigel was melted at 4°C and aliquoted for storage. Pipe tips were pre-cooled at 4°C. A 48-well plate was placed on an ice box, and 120 μl of Matrigel was poured into each well (10 wells). The plate was gently shaken to ensure even distribution of the Matrigel. The plate was then incubated at 37°C for 1 hour to allow the Matrigel to solidify. HUVEC cells of good condition (P6 generation or lower, purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) were digested and counted, with a cell density of 4 × 10⁶ cells / well. 5 HUVEC cells were plated at a density of 150 μl / ml into 48-well plates coated with matrix gel. VEGF (100-20-10UG, PeproTech) was added to each well at a final concentration of 50 ng / ml. After 10 min, prepared Cilengitide (HY16141, MCE) and PSFL-NK-13 (0, 2 nM, 20 nM, and 2 μM) were added. HUVEC cell tubule formation was observed at 6 h, 12 h, and 24 h. Images were taken of the central well under a 100× microscope, and the tubule counts were statistically analyzed.

[0091] like Figure 9 As shown, low concentrations of the αvβ3 inhibitor Cilengitide promote angiogenesis. HUVEC tubule formation assays showed that, compared to the control group (0.5% DMSO), Cilengitide at a concentration of 2 nM for 6 h significantly increased the number of tubules formed by endothelial cells, while higher concentrations inhibited tubule formation. The peptide PSFL-NK-13, at administration concentrations of 2 nM, 20 nM, and 2 μM, did not promote the formation of tubules, and high concentrations inhibited the formation of endothelial cell tubules.

[0092] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A specific interfering polypeptide, characterized in that, The amino acid sequence of the specific interfering polypeptide is shown in SEQ ID No. 1: SEQ ID No. 1: NMEAQNTTEVYVK.

2. The use of the specific interfering polypeptide according to claim 1 in the preparation of a tumor-treating drug, characterized in that, The tumor is either breast cancer or colorectal cancer.

3. The application of the specific interfering polypeptide of claim 1 in combination with CD47 antibody in the preparation of a tumor treatment drug, characterized in that, The tumor is either breast cancer or colorectal cancer.

4. The application according to claim 2 or 3, characterized in that, The drug comprises a specific interfering polypeptide with the amino acid sequence SEQ ID No. 1 and its pharmaceutically acceptable carrier or excipient.

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