Specific interference polypeptide and application thereof

Abstract

The invention discloses a specific interference polypeptide and application thereof. The amino acid sequence of the specific interference polypeptide disclosed by the invention is an amino acid sequence as shown in one of SEQ ID No.1 and SEQ ID No.1: NMEAQNTTEVYVK; sEQ ID No. 2: QNTTEVYVK is shown as SEQ ID No. 2. The specific interference polypeptide provided by the invention targets alpha v beta 3 / CD47-SIRP alpha for interaction, has no side effects of splitting erythrocytes and promoting tumor angiogenesis, and can be used for preparing medicines for treating tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a specific interfering polypeptide and application thereof. Background Art

[0002] CD47 (integrin-associated protein, IAP) belongs to the immune supermolecule family and is a transmembrane glycoprotein present 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 a variety of 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. The expression of CD47 is used by macrophages to distinguish "self" from "non-self" and can interact with signal regulatory protein α (SIRPα) on macrophages. The binding of CD47 to SIRPα promotes the phosphorylation of tyrosine residues in two typical immunoreceptor tyrosine-based inhibitory moieties (ITIMs) encoded in the cytoplasmic tail of SIRPα. Subsequently, the phosphorylated ITIM functions to recruit and activate protein tyrosine phosphatases (PTpases), particularly the Src homology 2 (SH2) domain-containing phosphatases SHP-1 and SHP-2. The interaction of the phosphatase SH2 domain with the phosphorylated ITIM of SIRPα disrupts their autoinhibitory activity on the pTpase domain, thereby triggering enzyme activity. SIRPα also binds to SH2 domain-containing tyrosine phosphatases, both of which 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. It is also a new signal used by tumor cells to evade detection and destruction.

[0003] In multicellular organisms, cell adhesion to the surrounding extracellular matrix (ECM) is crucial for the physiological development and functional integrity of tissues. Integrins are ubiquitous heterodimeric transmembrane glycoprotein adhesion receptors that are involved in almost every step from primary tumors to metastatic tumors through their multifaceted roles as signaling molecules, mechanical sensors, and key components of cell migration mechanisms. The ability of integrins to determine the responsiveness of cells to various inputs stems from their ability to differentially recognize different environments. To achieve this flexibility, each integrin is composed of an α subunit and a β subunit, of which 18 α subunits and 8 β subunits are combined to form 24 heterodimers with different known functions in a stable non-covalent connection. According to different types of ECM components, integrins can be divided into two major categories: receptors that recognize the Arg-Gly-Asp (RGD) peptide motif and receptors that are independent of the RGD binding region. Eight integrins that recognize the RGD motif (αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, α8β1, and αIIbβ3) constitute the most important integrin receptor subfamily and are involved in tumorigenesis and metastasis. Blocking integrin signals 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 affected by a variety of factors and change, thereby being more widely involved in various physiological disease processes. Among them, integrin αvβ3 is the most well-studied integrin involved in tumor progression, especially in tumor angiogenesis. The use of antibodies (taracizumab, Abituzumab and Intetumumab, etc.), peptides or peptidomimetics (cyclopeptide Cilengitide) and other small molecule antagonists to inhibit αvβ3 signaling has certain potential in tumor treatment, delivery of anti-tumor agents or imaging of tumor lesions (PET tracers based on RGD peptides), but there is also a side effect that low concentrations of αvβ3 anti-tumor preparations promote tumor angiogenesis. Although small molecules and peptides targeting integrins are currently used to treat fibrosis and ocular vascular diseases, there are still many problems to be solved in terms of safety and efficacy of anti-tumor agents targeting the αv series integrins (especially αvβ3, αvβ5).

[0004] In many tumor types, αvβ3 and CD47 are highly expressed. CD47 and αvβ3 are used as separate drug targets, but the development of anti-tumor drugs is not smooth. αvβ3 actively regulates the expression of PD-L1 in the tumor microenvironment and is therefore an important component of immune escape; the loss of αvβ3 inhibits the growth of primary tumors in mice, while most tumor patients currently receiving anti-PD-1 or anti-PD-L1 treatment in the clinic are unresponsive to treatment, and targeted αvβ3 treatment may make tumors more sensitive to the destruction of this axis, suggesting that in addition to PD-1 or PD-L1, there are more unknown mechanisms for αvβ3 to participate in tumor immune escape. Red blood cells express CD47 but not αvβ3; in vitro protein immunoprecipitation also proves that αvβ3 and CD47 can interact directly. These preliminary results lead us to raise related questions, such as: whether αvβ3 and CD47, which are highly expressed on the surface of tumor cell membranes, interact directly and produce functional associations; whether this interaction is static or dynamic; and whether αvβ3 is in an activated or inactivated state. This also leads to a bolder conjecture: if αvβ3 coexisting with CD47 can regulate the function of the CD47-SIRPα immune checkpoint, the immune checkpoint therapy that specifically interferes with CD47 / αvβ3 on the surface of cancer cells will not affect the function of red blood cells (red blood cells only express CD47), thereby eliminating the hemolytic effect of CD47 antibodies on red blood cells and the effect of SIRPα antibody Fc effect on myeloid cells (macrophages, monocytes). In addition, specific interference with the CD47 / αvβ3 interaction interface on the surface of cancer cells will also eliminate the side effects of interfering with the binding of CD47 to other ligands, such as thrombospondin-1 (TSP-1) vascular endothelial growth factor receptor 2 (VEGFR2), CD36 and Fas (CD95), or the effect of directly inhibiting αvβ3 on its normal function of recognizing ECM proteins containing the RGD tripeptide motif.

[0005] In a variety of tumor cell lines (especially the triple-negative breast cancer cell line MDA-MB-231) and 151 clinical samples of breast cancer and its adjacent or normal tissues, CD47 and ITGαvβ3 were found to have high expression in correlation with each other. Reducing the membrane expression of either of them on tumor cells can significantly reduce the distribution of the other on the membrane, thereby weakening the CD47-SIRPα axis and increasing the phagocytosis of cancer cells by macrophages, thereby significantly inhibiting tumor growth.

[0006] CD47-signal regulatory protein (SIRP) α signaling can enable malignant cells to evade macrophage-mediated phagocytosis, so inhibiting the CD47-SIRPα signaling axis represents a promising strategy for treating cancer. However, this treatment method, which has entered clinical trials, has a series of problems, including anemia, unclear upstream and downstream signaling mechanisms, different effects on different tumors, and promotion of tumor angiogenesis. Summary of the invention

[0007] Purpose of the invention: The purpose of the present invention is to provide a specific interfering polypeptide and its application. The specific interfering polypeptide provided by the present invention targets the αvβ3 / CD47-SIRPα interaction, has no side effects of lysing red blood cells and promoting tumor angiogenesis, and can be used to prepare drugs for treating tumors.

[0008] CD47 / αvβ3 forms a mutually stable complex on the cancer cell surface through direct interaction. This state can be simultaneously inhibited by CD47 antibodies, αvβ3 activating antibodies, αvβ3 inhibitory peptides Cyclo (-RGDfk) and Cilengitide, and extracellular Ca 2+ , and knockdown of Talin, a key protein in αvβ3 activation, thereby reducing CD47 and increasing the phagocytosis of macrophages, suggesting that activated αvβ3 may interact with CD47; however, this state cannot be interfered with by the αvβ3 inhibitor RGDS. The αvβ3 extracellular ligand fibronectin can increase the distribution of the complex on the membrane and can also be affected by αvβ3 non-activating antibodies, which shows that the αvβ3 interacting with CD47 is independent of the simple activation or inactivation state. One or more special conformations.

[0009] The present invention studies the interaction mechanism of αvβ3 / CD47-SIRPα and finds that it can target αvβ3 / CD47

[0010] -SIRPα-specific interference peptides. These peptides can reduce the expression of CD47 and αvβ3, thereby increasing the phagocytic effect of macrophages on tumors, and ultimately play a role in treating tumors. At the same time, it is important that these compounds have no side effects of lysing red blood cells and promoting tumor angiogenesis.

[0011] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0012] The present 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 interfering polypeptide of the present invention is prepared according to the prior art.

[0016] The present invention also provides the use of the specific interfering polypeptide in the preparation of drugs for treating tumors.

[0017] The present invention also provides the use of the specific interfering polypeptide in combination with CD47 antibody in the preparation of a drug 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. 1-2 and a pharmaceutically acceptable carrier or excipient thereof.

[0020] The auxiliary materials include one or more of emulsifiers, solubilizers, antioxidants, wetting agents, diluents, preservatives, disintegrants, binders or lubricants.

[0021] The dosage form of the drug is tablet, granule, injection, capsule, oral liquid or pill.

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

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

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

[0025] The specific interfering polypeptide reduces the expression of CD47 and αvβ3 and enhances the effect of macrophages phagocytizing tumor cells at 0.5-5 μM.

[0026] When the specific interfering polypeptide is used in combination with the CD47 antibody at a concentration of 0.5-5 μM, the effect of the CD47 antibody in promoting macrophages to phagocytize tumor cells is enhanced.

[0027] The specific interfering polypeptide has no lytic effect on red blood cells at 0.125-32 μM.

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

[0029] The specific interfering polypeptide reduces tumor volume at 0.5-8 mg / kg.

[0030] Beneficial effects:

[0031] The specific interfering polypeptides provided by the present invention can significantly reduce the membrane expression of CD47 and αvβ3 in tumor cells, increase the phagocytosis of macrophages, slow down the growth rate of breast cancer transplanted in situ in immunodeficient and normal mice, and are effective against a variety of other tumors such as small cell lung cancer, colorectal cancer, and glioma. Most importantly, these polypeptides have no side effects of lysing red blood cells and promoting tumor angiogenesis. Therefore, the specific interfering polypeptides of the present invention can be used to prepare drugs for treating tumors. In addition, the effect of the specific interfering polypeptides of the present invention in combination with CD47 antibodies is stronger than the effect of using either one alone. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0034] Figure 3 The specific peptide PSFL-NK-13 combined with CD47 antibody can enhance the promotion effect of CD47 antibody (B6H12) on macrophage phagocytosis of tumor cells. n=3, ***p<0.001vs CD47 antibody (B6H12) group. Figure 4 The specific peptide QK-9 reduces CD47 ( Figure 4 A) and αvβ3( Figure 4 B) and increased the phagocytosis of MDA-MB-231 by macrophages ( Figure 4 C); n = 3-4, * p<0.05vs 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 membrane of HL-60 cells 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 breast cancer orthotopic tumors in nude mice; Figure 6 A is the statistical graph of tumor volume-time curves of the Ctrl group and three dose 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 three dose groups of PSFL-NK-13 (0.5, 2 and 8 mg / kg); Figure 6 C is the statistical graph of tumor weight in the Ctrl group and three dose 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 inhibits the progression of colorectal cancer tumors in nude mice; Figure 7 A is the statistical graph of tumor volume-time curves of Ctrl group and PSFL-NK-13 (2 mg / ml) group; Figure 7 B is a schematic diagram of tumor size in the Ctrl group and the PSFL-NK-13 (2 mg / kg) group; Figure 7 C is the statistical graph of tumor weight in the Ctrl group and the PSFL-NK-13 (2 mg / kg) group; n=6, *** p<0.001vsCtrl.

[0038] Figure 8 The figure is a comparison of the hemolytic effects of polypeptide PSFL-NK-13 and antibody B6H12 on RBCs.

[0039] Fig. 9Shown are the effects of polypeptides PSFL-NK-13 and Cilengitide on HUVEC tubule formation and statistical graphs. DETAILED DESCRIPTION

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

[0041] The reagents or instruments used in the present invention without indicating the manufacturer are all conventional products that can be purchased on the market.

[0042] Example 1 Preparation of specific interfering polypeptide PSFL-NK-13

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

[0044] Synthesize peptides using solid phase peptide synthesis technology: weigh 5g Fmoc-aa-Wang resin (Gill Biochemical), add 10 times the mass of dichloromethane to swell for 20 minutes, add 1.58ml deprotection solution (morpholine: N, N-dimethylformamide = 6:4, v / v) and react for 30min. After the reaction, wash the resin alternately with dichloromethane and N, N-dimethylformamide 3 times. Weigh Fmoc-amino acid (8.5mmol, Jizhi Biochemical) and O-(7-azabenzotriazole-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU, 10.2mmol, Aladdin) and dissolve them in N, N-dimethylformamide (10.2mmol), mix well, and add the reaction solution to the resin to react for 4h. After the reaction, wash the resin alternately with dichloromethane and N, N-dimethylformamide 3 times. Dry with nitrogen flow. After drying, 200 mL of cutting solution (trifluoroacetic acid-deionized water-triisopropylsilane = 95.0:2.5:2.5, v / v) was added to the resin and shaken for 1.5 h to cut the target polypeptide from the resin. The target polypeptide was precipitated with icy ether, the crude peptide was collected by centrifugation, and purified by HPLC.

[0045] The purity of the purified polypeptide was identified by 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 automatic amino acid sequencer.

[0046] The purity of PSFL-NK-13 peptide is more than 95%;

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

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

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

[0050] Synthesis of peptides using solid phase peptide synthesis technology: Synthesis of peptides using solid phase peptide synthesis technology: Weigh 5g Fmoc-aa-Wang resin (Gill Biochemical), add 10 times the mass of dichloromethane to swell for 20 minutes, add 1.58ml deprotection solution (morpholine: N, N-dimethylformamide = 6:4, v / v) and react for 30min. After the reaction, wash the resin alternately with dichloromethane and N, N-dimethylformamide 3 times. Weigh Fmoc-amino acid (8.5mmol, Jizhi Biochemical) and O-(7-azabenzotriazole-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU, 10.2mmol, Aladdin) and dissolve them in N, N-dimethylformamide (10.2mmol), mix well, and add the reaction solution to the resin to react for 4h. After the reaction, wash the resin alternately with dichloromethane and N, N-dimethylformamide 3 times. Dry with nitrogen flow. After drying, 200 mL of cutting solution (trifluoroacetic acid-deionized water-triisopropylsilane = 95.0:2.5:2.5, v / v) was added to the resin and shaken for 1.5 h to cut the target polypeptide from the resin. The target polypeptide was precipitated with icy ether, the crude peptide was collected by centrifugation, and purified by HPLC.

[0051] The purity of QK-9 peptide is more than 95%;

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

[0053] Example 3 Specific interfering polypeptide PSFL-NK-13 reduces the expression of CD47 and αvβ3 on breast cancer cells MDA-MB-231 and enhances the phagocytosis of macrophages

[0054] 1. Detection of the expression of CD47 and αvβ3 on the surface of MDA-MB-231 cells by flow cytometry

[0055] Experimental methods:

[0056] MDA-MB-231 cells (purchased from ATCC) were inoculated and cultured overnight in a 37°C incubator until the cells adhered well. 4 μM PSFL-NK-13 or blank solvent (0.5% DMSO) was added for 48 h, and the cells were transfected with siRNA (Shanghai GenePharmaCo Ltd.). After changing the medium, the cells were cultured in the incubator for another 48 h. The upper layer of the culture medium was discarded, and the cell suspension was resuspended after digestion with trypsin (Gibco), sucked into a 5 ml flow tube, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, 1 ml PBS was added, and the cells were washed once, and centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was carefully discarded, and the cells were resuspended in 100 μl PBS; take out the flow cytometry antibodies FITC-CD51 / 61 (304402, Biolegend) and APC-CD47 (17-0479-42, invitrogen) and put them on an ice box, and incubate them in a shaker at room temperature for 30 minutes to allow the cells to be fully labeled with the antibodies. The shaker speed is 150 rpm; add 500 μl PBS to resuspend the cells, protect them from light and place them on ice; use a flow cytometer (BD LSR-Fortessa) to collect the cells, and the data are analyzed using FlowJo 10.6 software.

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

[0058] 2. Flow cytometry to measure macrophage phagocytosis of tumor cells

[0059] Experimental methods:

[0060] THP-1 cells (from ATCC) were inoculated in 24-well plates and induced into macrophages by adding Phorbol 12-Myristate 13-Acetate (PMA, from MCE) at a final concentration of 200 ng / ml. MDA-MB-231 cells (from ATCC) were treated with blank solvent (0.5% DMSO) or 4 μM PSFL-NK-13 for 48 h, then trypsinized and counted, and the cell density was adjusted to 2×10 5500 μl of cell suspension was placed in an EP tube, centrifuged at 1000 rpm for 5 min, and the cells were resuspended in 500 μl of RPMI-1640 basic culture medium (C22400500BT, Gibco); fluorescent probe Protonex was added TM Red 600 (21207, AAT Bioquest) was prepared with a concentration of 5mM stock solution, added to the cell suspension to a final concentration of 0.5μM, and incubated at 37°C in a cell culture incubator for 30min. After adding RPMI-1640 to the EP tube, centrifuged at 1000rpm for 5min, and then resuspended with RPMI-1640 for use. The THP-1 cell supernatant was removed, and the above-mentioned MDA-MB-231 cell suspension was added and incubated together for 1h; the cell supernatant was removed, and the cells were digested with trypsin, centrifuged at 1000rpm for 5min, and the cells were washed once with PBS solution, and the cells were resuspended in 500μl of PBS. The phagocytosis of macrophages (THP-1) on tumor cells (MDA-MB-231) was detected by flow cytometry.

[0061] The results of flow cytometry analysis showed that: Figure 2 As shown, compared with the control group (DMSO), the proportion of tumor cells phagocytosed by THP1 cells increased significantly after 48 hours of action of 4 μM peptide PSFL-NK-13, and there was a significant difference between the two groups. The phagocytic proportion of the PSFL-NK-13 administration group increased from 9.1±1.2% to 31.5±4.3%.

[0062] Example 4 The specific interfering polypeptide PSFL-NK-13 is used in combination with CD47 antibody to enhance the effect of CD47 antibody in promoting macrophage phagocytosis of MDA-MB-231 cells

[0063] THP-1 cells (derived from ATCC) were inoculated in 24-well plates, and phorbol ester (Phorbol 12-Myristate 13-Acetate, PMA, derived from MCE) was added at a final concentration of 200 ng / ml to induce them into macrophages. MDA-MB-231 cells (derived from ATCC) were taken separately, and blank solvent (0.5% DMSO), 4 μM PSFL-NK-13, 10 μg / ml CD47 antibody (B6H12, Abcam), 4 μM PSFL-NK-13 + 10 μg / ml CD47 antibody were used for 48 hours, and then digested with trypsin, counted, and the cell density was adjusted to 2×10 5500 μl of cell suspension was placed in an EP tube, centrifuged at 1000 rpm for 5 min, and the cells were resuspended in 500 μl of RPMI-1640 basic culture medium (C22400500BT, Gibco); fluorescent probe Protonex was added TM Red 600 (21207, AAT Bioquest) was prepared with a concentration of 5mM stock solution, added to the cell suspension to a final concentration of 0.5μM, and incubated at 37°C in a cell culture incubator for 30min. After adding RPMI-1640 to the EP tube, centrifuged at 1000rpm for 5min, and then resuspended with RPMI-1640 for use. The THP-1 cell supernatant was removed, and the above-mentioned MDA-MB-231 cell suspension was added and incubated together for 1h; the cell supernatant was removed, and the cells were digested with trypsin, centrifuged at 1000rpm for 5min, and the cells were washed once with PBS solution, and the cells were resuspended in 500μl of PBS. The phagocytosis of macrophages (THP-1) on tumor cells (MDA-MB-231) was detected by flow cytometry.

[0064] The results of flow cytometry analysis showed that: Figure 3 As shown, compared with the CD47 antibody (B6H12) acting alone, the proportion of tumor cells phagocytosed by THP1 cells increased significantly after 48 hours of combined action of 4μM polypeptide PSFL-NK-13 and CD47 antibody (B6H12), and there was a significant difference between the two groups. The phagocytic ratio increased from 27.1±2.4% in the CD47 antibody (B6H12) alone administration group to 48.7±4.4% in the combined administration group.

[0065] Example 5 Specific interfering polypeptide QK-9 reduces the expression of CD47 and αvβ3 on breast cancer cells MDA-MB-231 and enhances the phagocytosis of macrophages

[0066] 1. Detection of the expression of CD47 and αvβ3 on the surface of MDA-MB-231 cells by flow cytometry

[0067] Experimental methods:

[0068] MDA-MB-231 cells were inoculated and cultured in a 37°C incubator overnight until the cells adhered well to the wall; the cells were transfected with siRNA, and the culture medium was changed and continued to be cultured in the incubator for 48 hours; the upper layer of the culture medium of the cells was aspirated, the cell suspension was resuspended after trypsin digestion, and aspirated into a 5 ml flow tube, and centrifuged at 1000 rpm for 5 minutes; the supernatant was discarded, 1 ml PBS was added, and the cells were washed once, and centrifuged at 1000 rpm for 5 minutes; after centrifugation, the supernatant was carefully aspirated and the cells were resuspended in 100 μl PBS; the flow antibodies FITC-CD51 / 61 and APC-CD47 were taken out and placed on an ice box, and incubated in a shaker at room temperature for 30 minutes to fully label the cells with antibodies, and the shaker speed was 150 rpm; 500 μl PBS was added to resuspend the cells, and the cells were placed on ice in the dark; the cells were collected using a flow cytometer (BD LSR-Fortessa), and the data were analyzed using FlowJo 10.6 software.

[0069] 2. Flow cytometry to measure macrophage phagocytosis of tumor cells

[0070] Experimental methods:

[0071] THP-1 cells were seeded in 24-well plates and PMA was added at a final concentration of 200 ng / ml to induce them into macrophages. After 48 h of exposure to blank solvent (0.5% DMSO) or 4 μM QK-9, the cells were digested with trypsin and counted. The cell density was adjusted to 2×10 5 500 μl of cell suspension was placed in an EP tube, centrifuged at 1000 rpm for 5 min, and the cells were resuspended in 500 μl of RPMI-1640 basic medium; the fluorescent probe Protonex was added TM Red 600 (AAT Bioquest) was prepared with a stock solution of 5 mM, added to the cell suspension to a final concentration of 0.5 μM, and incubated for 30 min. After adding RPMI-1640 to the EP tube, the tube was centrifuged at 1000 rpm for 5 min, and the cells were resuspended in RPMI-1640. The supernatant was removed, and the MDA-MB-231 cell suspension was added and incubated together. The cell supernatant was removed, and the cells were digested with trypsin, centrifuged at 1000 rpm for 5 min, and the cells were washed once with PBS solution, and the cells were resuspended in 500 μl of PBS. The phagocytosis of tumor cells by macrophages was detected by flow cytometry.

[0072] Flow cytometry results: Figure 4As shown, compared with the control group Ctrl (DMSO), 4 μM peptide QK-9 reduced the expression of integrin αvβ3 and CD47 on the MDA-MB-231 cell membrane after 48 hours of action. The proportion of tumor cells phagocytosed after QK-9 treatment increased significantly, and there was a 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 Specific interference peptide PSFL-NK-13 on the expression of CD47 and αvβ3 on colorectal cancer HCT-116 cells, lung cancer A549 cells and leukemia HL-60 cells

[0074] Flow cytometry was used to detect the expression of CD47 and αvβ3 on the surface of colorectal cancer HCT-116 cells, lung cancer A549 cells and leukemia HL-60 cells.

[0075] Rectal cancer HCT-116 cells, lung cancer A549 cells or leukemia HL-60 cells (all from ATCC) were inoculated and cultured overnight in a 37°C incubator until the cells adhered well. Blank solvent (0.5% DMSO) or 4μM PSFL-NK-13 was used for 48h, the upper layer of the cell culture medium was aspirated and discarded, the cell suspension was resuspended after trypsin digestion, aspirated into a 5ml flow tube, and centrifuged at 1000rpm for 5min; the supernatant was discarded, 1ml PBS was added, washed once, and centrifuged at 1000rpm for 5min; after centrifugation, the supernatant was carefully aspirated and the cells were resuspended in 100μl PBS; the flow antibodies FITC-CD51 / 61 and APC-CD47 were taken out and placed on an ice box, and incubated in a shaker at room temperature for 30min to fully label the cells with antibodies, and the shaker speed was 150rpm; 500μl PBS was added to resuspend the cells, and the cells were placed on ice in the dark; a flow cytometer (BD Biosciences) was used for analysis. The cells were collected by LSR-Fortessa, and the data were analyzed by FlowJo 10.6 software.

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

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

[0078] Example 7: Specific interfering peptide PSFL-NK-13 inhibits the development of breast cancer in situ tumors in nude mice

[0079] Test method:

[0080] The subcutaneous transplanted tumor model of nude mice (8 weeks old, Jiangsu Huachuang Xinnuo) was established by orthotopic injection of MDA-MB-231 cells. MDA-MB-231 cells (from ATCC) were cultured at 4×10 6 The number of inoculations per mouse was inoculated in the mammary fat pad of the mouse. Two weeks after the tumor cells were orthotopically transplanted, the mice were randomly divided into four groups according to the tumor growth, control group (Ctrl, normal saline), PSFL-NK-13 low-dose group (0.5 mg / kg), PSFL-NK-13 medium-dose group (2 mg / kg) and PSFL-NK-13 high-dose group (8 mg / kg). The peptide PSFL-NK-13 was used to treat mouse tumors at a frequency of local subcutaneous administration three times a week at a dose of 0.5 mg / kg, 2 mg / kg and 8 mg / kg, for a total of 4 weeks.

[0081] After 4 weeks of treatment, the tumor volumes of the three dose groups were significantly reduced, and the ex vivo tumors were photographed (see Figure 6 B) It was observed that the tumor volume of the drug-treated group was significantly reduced, and the tumor weight of the PSFL-NK-13 polypeptide-treated group was significantly reduced compared with the control group Ctrl (see 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] The nude mouse subcutaneous transplant tumor model was established by subcutaneous injection of HCT-116 cells. HCT-116 cells (from ATCC) were injected at a concentration of 1×10 6 The number of inoculations / mouse was inoculated in the subcutaneous position of the abdomen of nude mice (8 weeks old, Jiangsu Huachuang Xinnuo). One week after the tumor cells were subcutaneously transplanted, the nude mice were evenly randomly divided into control group (Ctrl, saline) and PSFL-NK-13 group (2 mg / kg) according to the tumor growth. The peptide PSFL-NK-13 was used at a dose of 2 mg / kg at a frequency of daily local subcutaneous administration to treat nude mouse tumors.

[0085] After three weeks of treatment, it was found that the tumor volume of nude mice treated with peptide PSFL-NK-13 continued to decrease. Compared with the untreated group, the tumor after peptide treatment was significantly reduced, and the tumor weight was significantly different. The tumor size after dissection was photographed and shown in the figure. The weight of the tumor in the Ctrl group was 0.61±0.11g, and the weight of the tumor in the PSFL-NK-13 (2mg / kg) group was 0.24±0.12g. There was a significant difference in the tumor weight between the two groups (see Figure 7 AC).

[0086] Example 9: No hemolysis of red blood cells by specific interfering polypeptide PSFL-NK-13

[0087] Blood was collected from healthy volunteers, treated with a sampling tube containing an anticoagulant, and mixed with human lymphocyte separation solution (Tianjin Haoyang Biological Company, LTS10771). The bottom layer of blood cells was separated and collected, resuspended with PBS, centrifuged at 400g for 10 minutes, and washed twice. Red blood cells were mixed with PBS to form a 2.5% (v / v) cell suspension, and then 50μl of red blood cell suspension was added to a 96-well round bottom plate and mixed with 50μl containing a concentration of 0.125-32μM PSFL-NK-13 polypeptide or a concentration of 0.3125-80μg / ml CD47 antibody (B6H12). The plate was incubated at 37°C for 4-6h, and the red floccules in the supernatant in the well were observed for blood cell agglutination, while the colorless supernatant and all red blood cell sedimentation indicated no significant changes. The middle field of view of the well plate was photographed at 100× under a white light microscope.

[0088] The red blood cell hemolysis experiment found that: Figure 8 As shown, CD47 antibody (B6H12) can cause agglutination of erythrocytes at a concentration of 5 mg / ml, resulting in hemolysis and lysis of erythrocytes, while polypeptide PSFL-NK-13 has no effect on erythrocytes at the corresponding administration concentration, and no hemolysis of erythrocytes occurs. Even when the administration concentration is increased to 100 μM and 200 μM, no damage to erythrocytes is observed.

[0089] Example 10 Specific interfering polypeptide PSFL-NK-13 does not promote angiogenesis

[0090] 10 mg / ml Matrigel matrix was melted in a 4°C refrigerator and then stored in aliquots. The pipette tip was precooled at 4°C. A 48-well plate was placed on an ice box. 120 μl of Matrigel matrix was pipetted and spread into the wells. There were 10 wells. The plate was gently shaken to spread the Matrigel evenly on the bottom of the plate. The plate was placed in a 37°C incubator for 1 hour to allow the Matrigel to solidify. HUVEC cells in good condition within the P6 generation (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) were taken and digested and counted. The cell density was 4×10 5 / ml, 150μl cell suspension was spread into a 48-well plate covered with matrix gel; cytokine VEGF (100-20-10UG, PeproTech) with a final concentration of 50ng / ml was added to each well, and after 10min, the prepared drugs Cilengitide (HY16141, MCE) and PSFL-NK-13 (0, 2nM, 20nM and 2μM) were added; the tubule formation of HUVEC cells was observed at 6h, 12h and 24h, and the field of view in the middle well was selected under a 100× microscope for photography and recording, and the tubule counting results were statistically analyzed.

[0091] like Fig. 9 As shown, low concentrations of Cilengitide, an inhibitor of αvβ3, promote angiogenesis. The results of the HUVEC tubule formation experiment showed that compared with the control group (0.5% DMSO), Cilengitide at a concentration of 2nM for 6 hours could significantly increase the number of tubules formed by endothelial cells, while at higher concentrations it could inhibit the formation of tubules. The polypeptide PSFL-NK-13 did not promote the formation of the number of tubules at the administration concentrations of 2nM, 20nM and 2μM, and high concentrations could inhibit the formation of endothelial cell tubules.

[0092] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present 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 the amino acid sequence shown in one of SEQ ID No. 1 to 2: SEQ ID No.1: NMEAQNTTEVYVK; SEQ ID No. 2: QNTTEVYVK.

2. Use of the specific interfering polypeptide according to claim 1 in the preparation of drugs for treating tumors.

3. Use of the specific interfering polypeptide according to claim 1 in combination with CD47 antibody in the preparation of drugs for treating tumors.

4. The use according to claim 2 or 3, characterized in that: The tumor is breast cancer, small cell lung cancer, colorectal cancer, glioma or leukemia.

5. The use according to claim 2 or 3, characterized in that: The drug comprises a specific interfering polypeptide represented by one of the amino acid sequences SEQ ID No. 1-2 and a pharmaceutically acceptable carrier or excipient thereof.

6. The use according to claim 2 or 3, characterized in that: The specific interfering polypeptide reduces the expression of CD47 and αvβ3 and enhances the effect of macrophages phagocytizing tumor cells at 0.5-5 μM.

7. The use according to claim 3, characterized in that: When the specific interfering polypeptide is used in combination with the CD47 antibody at a concentration of 0.5-5 μM, the effect of the CD47 antibody on promoting macrophages to phagocytize tumor cells is enhanced.

8. The use according to claim 2 or 3, characterized in that: The specific interfering polypeptide has no lytic effect on red blood cells at 0.125-32 μM.

9. The use according to claim 2 or 3, characterized in that: The specific interfering polypeptide does not promote tumor angiogenesis at 2 nM-2 μM.

10. The use according to claim 2 or 3, characterized in that: The specific interfering polypeptide reduces tumor volume at 0.5-8 mg / kg.

Images