CN110981942B - A polypeptide RS-17 with anti-CD47 immune checkpoint antagonistic activity and its application - Google Patents

Abstract

The invention discloses a polypeptide RS-17 with anti-CD47 immune checkpoint antagonistic activity and an application thereof, belonging to the field of biomedicine. The polypeptide sequence of the present invention is RRYKQDGGWSHWSWPWS-NH ₂ . This polypeptide can specifically recognize CD47 on the surface of tumor cells, and has the ability to block the binding of CD47 to its corresponding ligand, thereby blocking the autologous signal generated by the interaction between CD47 and SIRPa on the surface of macrophages. Thereby eliminating the immunosuppressive effect of the tumor and achieving the effect of specifically killing the tumor.

Description

A polypeptide RS-17 with anti-CD47 immune checkpoint antagonistic activity and its application

technical field

The invention belongs to the field of biological medicine, and more particularly relates to a polypeptide capable of blocking the binding of CD47 on the surface of tumor cells and its corresponding ligand and its application.

Background technique

CD47 (Cluster of Differentiation 47), also known as integrin-associated protein (IAP), is a widely expressed glycoprotein with five transmembrane domains, encoded by the CD47 gene in humans, and plays an important role in self-signaling effect. It belongs to the immunoglobulin superfamily and can interact with integrin (Integrin), thrombospondin (TSP-1) and signal regulatory protein alpha (SIRPα), among which the interaction between CD47 and signal regulatory protein alpha (SIRPα) is studied as the most in-depth part. Bidirectional signal transduction mediated by its corresponding receptors results in different intercellular responses, including inhibition of phagocytosis, cell fusion, and T cell activation.

CD47 is widely expressed on the surface of many healthy cells, and releases a "Don't eat me" signal by binding to SIRPα on the surface of macrophages, telling macrophages not to engulf them, thereby protecting healthy cells from being destroyed by the immune system, while When cells age or become diseased, CD47 is gradually lost on the cell surface to help the body recognize and dispose of those cells. Unfortunately, this mechanism is exploited by cancer cells. The current study found that tumor cells can escape the attack of macrophages by expressing CD47. In order to protect themselves, cancer cells arrange more CD47, CD47 and SIRPα on the surface than normal cells. Binding also releases a "Don't eat me" signal, and macrophages do not phagocytose tumor cells.

The blockade of CD47 by monoclonal antibodies can trigger the phagocytosis of various cancer cells by macrophages in vitro. The principle is that the highly expressed CD47 on the surface of cancer cells combines with the SIRPa receptor on the surface of macrophages to generate a corresponding autologous signal (Don't eat me signal), thereby inhibiting the macrophages targeting tumor cells. Phagocytosis and antigen presentation. Therefore, it has become a new direction for cancer treatment to block the inhibition of phagocytosis by the autologous signal generated by the interaction of CD47-SIRPa, so that the immune system can normally remove abnormal cancerous cells and realize its normal function.

At present, there are no drugs for CD47-targeted therapy that have been officially launched. The two related drugs with the fastest progress are Hu5F9-G4 from Forty Seven, a subsidiary of Stanford University, and TI-061 from Tioma, a subsidiary of the University of Washington. Both drugs have entered Phase II clinical trials. And the two drugs are significantly effective against non-Hodgkin's lymphoma when used in combination with the anti-CD47 inhibitor Lipitorumab, far better than Lipitorumab alone. However, the use of two CD47 mAbs alone is less effective than Lipitorumab alone in the treatment of non-Hodgkin's lymphoma. However, both drugs belong to the monoclonal antibody class, and there is no research report on any small molecule drugs or peptide drugs.

Therefore, CD47, as a signaling molecule that prevents phagocytosis by macrophages, has potential as a therapeutic target for certain cancers.

SUMMARY OF THE INVENTION

1. The problem to be solved

The present invention provides a polypeptide with immune checkpoint antagonistic activity and its application. Currently, there is no official marketed anti-tumor drug targeting CD47 for targeted therapy, but the polypeptide of the present invention acts on CD47 to inhibit CD47 and SIRPα. The pathway is highly innovative to achieve tumor suppressor effect. It has been verified by various experimental models that it has a good tumor-inhibiting effect, so it has great development prospects.

2. Technical solutions

In order to solve the above problems, the technical scheme adopted in the present invention is as follows:

A polypeptide RS-17 with anti-CD47 immune checkpoint antagonistic activity is characterized in that its amino acid sequence is: RRYKQDGGWSHWSPPWSS-NH ₂ and an amino acid sequence that is more than 80% homologous to the polypeptide.

"-NH ₂ " in the described polypeptide RS-17 sequence indicates that the C-terminal of the peptide chain has been modified by amidation.

The application of the polypeptide RS-17 with anti-CD47 immune checkpoint antagonistic activity in preparing a drug for preventing or treating cancer.

The application is characterized in that the cancer includes: epithelial tissue carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer, peritoneal carcinoma, hepatocellular carcinoma, gastric or stomach cancer, pancreas cancer, gallbladder cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer.

A pharmaceutical composition, characterized in that: the composition contains the polypeptide RS-17 described in 1 and one or more pharmaceutically acceptable excipients; wherein the excipients include conventional diluents in the pharmaceutical field, fillers agents, binders, wetting agents, absorption enhancers, surfactants, lubricants and stabilizers.

The pharmaceutical composition is characterized in that: the pharmaceutical composition is made into injection and dry powder injection. At present, the main immune checkpoint inhibitors in clinical practice are monoclonal antibodies, but the use of antibodies has problems such as large side effects and high treatment costs. Therefore, on the basis of analyzing a large number of traditional structures and pharmacological experiments, the self-designed polypeptide structure of the present invention has a good tumor suppressing effect.

definition

As used herein, the terms "a" or "an" may mean one.

As used herein, the term "or" means "and/or" unless expressly indicated that only the alternatives or the alternatives are mutually exclusive, although the present disclosure supports references to the alternatives only as well as "and/or" definition.

As used herein, the term "another" refers to at least a second or more.

As used herein, the term "about" is used to indicate that a value includes inherent variation in the error of the equipment, method used to determine the value, or variation that exists between subjects.

Cancers for which this method of treatment can be used include any malignant cell type, such as those found in solid tumors or hematological tumors. Generally, a tumor refers to a malignant or potentially malignant neoplasm or tissue mass of any size including primary and secondary tumors. A solid tumor is an abnormal mass or growth of tissue that usually does not contain cysts or fluid. Exemplary solid tumors may include, but are not limited to, tumors selected from the group consisting of: pancreas, gallbladder, colon, cecum, stomach, brain, head, neck, ovary, testis, kidney, larynx, sarcoma, lung, bladder, Melanoma, prostate and breast. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myeloma, and the like. Other examples of cancers that can be treated using the methods provided herein include, but are not limited to, carcinomas of epithelial tissue (carcinomas), lymphomas, blastomas, sarcomas, leukemias, squamous cell carcinomas, lung cancers (including small cell lung cancers, non-small cell lung cancers) , lung adenocarcinoma and lung squamous cell carcinoma), peritoneal carcinoma, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal carcinomas), pancreatic cancer, gallbladder cancer, glioblastoma, Cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, various head and neck cancers , melanoma, superficial diffuse melanoma, lentigo malignant melanoma, acral lentiginous melanoma, nodular melanoma, and B-cell lymphomas (including low-grade/follicular non-Hodgkin lymphoma) (NHL), small lymphocyte (SL) NHL, intermediate/follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphocytic NHL, high-grade small non-lysing cell NHL, bulky NHL, mantle cell lymphoma , AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, multiple myeloma, acute myeloid leukemia (AML), and chronic myelogenesis cell leukemia.

Cancers may specifically have, but are not necessarily limited to, the following histological types: neoplastic, malignant; epithelial carcinoma; epithelial carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatric carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma ; Trabecular adenocarcinoma; Adenoid cystic carcinoma; Adenoma in adenomatous polyps; Adenocarcinoma, familial polyposis colon; Solid carcinoma; Carcinoid tumor, malignant; Branch alveolar adenocarcinoma; Papillary adenocarcinoma; Cell carcinoma; Oncocytic carcinoma; Oncocytic adenocarcinoma; Basophilic carcinoma; Clear cell adenocarcinoma; Granular cell carcinoma; Follicular adenocarcinoma; Papillary and follicular adenocarcinoma; Non-cystic sclerosing carcinoma; Adrenal cortex carcinoma; endometrial carcinoma; skin adnexa carcinoma; apocrine gland adenocarcinoma; sebaceous adenocarcinoma; Cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous Epithelial metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; osteoblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; Lipcytoma, malignant; Paraganglioma, malignant; Extramammary paraganglioma, malignant; Pheochromocytoma; Globosarcoma, malignant melanoma; Pigmented nevus, malignant melanoma; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma ; Interstitial sarcoma; Mixed tumor, malignant; Mueller mixed tumor; Wilms tumor; Hepatoblastoma; Carcinosarcoma; Stromal tumor, malignant; Bladder tumor, malignant; Thyroid tumor, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; ovarian thyroid tumor, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma, hemangioendothelioma, malignant; cell tumor, malignant; lymphangioma, osteoma, concurrent osteosarcoma; chondrosarcoma; chondrocytoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's tumor; odontogenic tumor, malignant; ameloblastoma Cytodontosarcoma; Ameloblastoma, malignant; Ameloblastoma fibrosarcoma; Pineal tumor, malignant; Chordoma; Glioma, malignant; Ependymoma, Astrocytoma; Protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma Blastoma; Retinoblastoma; Olfactory Neurogenic Tumor; Meningioma, Malignant; Neurofibrosarcoma; Schwannoma, Malignant; Granular Cell Tumor, Malignant; Lymphoma Malignant; Hodgkin's Disease ; Paragranuloma; Malignant lymphoma, small lymphocytes; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Other specified non-Hodgkin's lymphoma; hyperplasia; multiple myeloma; mast cell sarcoma; immunoproliferative intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythrocytic leukemia; lymphosarcoid leukemia; myeloid leukemia; basophilic leukemia; eosinophilic Leukemia; monocytic leukemia; mast cell leukemia; megakaryocyte leukemia; myelosarcoma and hairy cell leukemia.

The cancer to be treated is preferably positive for CD47.

3. Beneficial effects

Compared with the prior art, the beneficial effects of the present invention are:

(1) At this stage, there is no official marketed tumor therapy drug targeting CD47 immune checkpoint, and the invention is highly innovative.

(2) The polypeptide of the present invention has a simple structure, is easy to synthesize, isolate and purify, effectively inhibits the CD47/SIRPα pathway, and eliminates the immunosuppressive effect;

(3) The adverse reactions and toxic side effects of the polypeptide of the present invention are weak;

(4) The polypeptides involved in the present invention have good immunosuppressive effects in in vitro and in vivo models, and the specific effects are to significantly increase the activity of macrophages to eliminate immunosuppressive effects, accelerate the removal of tumor cells, and produce tumor suppressive effects.

Description of drawings

Figure 1---The mass spectrometry detection report of the synthetic polypeptide RS-17.

Figure 2---High performance liquid chromatography detection report of synthetic polypeptide RS-17.

Figure 3 - Thermophoretic amplitude results between CD47-RS-17.

Figure 4 --- Western blot detection of tumor cell lines with high expression of CD47 and lymphocyte cell lines with high expression of SIRPα as positive cells, Figure A is a tumor cell line with high expression of CD47, and Figure B is a lymphocyte cell line with high expression of SIRPα .

Figure 5---Flow cytometry detects the binding of SCC-13 cells to FITC-conjugated anti-CD47 polypeptide.

Figure 6---Flow cytometry detects the binding of HepG2 cells to FITC-conjugated anti-CD47 polypeptide.

Figure 7--The results of co-incubating CFSE dye-labeled HepG2 and THP-1 macrophages for 2 hours at a polypeptide concentration of 20ug/ml with a 10x objective.

Figure 8--The results of co-incubation of CFSE dye-labeled HepG2 and RAW264.7 macrophages for 2 hours at a peptide concentration of 20ug/ml with a 10x objective.

Figure 9--Statistical results of phagocytosis index under the conditions of THP-1 and RAW264.7 macrophages, Figure A is the statistical results of THP-1 cells, Figure B is the statistical results of RAW264.7 cells

Figure 10--The changes in body weight and tumor volume of nude mice within seven days after the tumor cells pretreated with polypeptides were implanted in the armpits of nude mice. Figure A is the statistical data of body weight of nude mice, and Figure B is the statistical data of tumor volume of nude mice.

Figure 11---After the tumor cells pretreated with polypeptide were implanted into nude mice, the mean fluorescence intensity of GFP in nude mice on the seventh day

Figure 12 - Changes in body weight and tumor volume of nude mice treated with polypeptide within 21 days. Figure A is the statistical data of body weight of nude mice, and Figure B is the statistical data of tumor volume of nude mice.

Figure 13 --- Changes in relative tumor volume and relative tumor proliferation rate in nude mice treated with polypeptide within 21 days. Panel A is the relative tumor volume statistics, and Panel B is the relative tumor proliferation rate statistics.

Figure 14---Mean fluorescence intensity of GFP in vivo in nude mice treated with polypeptide on day 21

Detailed ways

The present invention will be further described below with reference to specific embodiments. The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person skilled in the art may use the technical contents disclosed above to change into equivalent embodiments with equivalent changes.

The polypeptide RS-17 designed in the present invention was synthesized by GenScript.

Embodiment 1RS-17 synthetic product quality detection

The synthetic RS-17 polypeptide has the sequence: RRYKQDGGWSHWSPPWSS-NH ₂ . Character information such as relative molecular mass, yield and purity were measured by mass spectrometry (Mass Spectrometry, MS) and high performance liquid chromatography (High Performance Liquid Chromatography\HPLC).

The specific chromatographic conditions are:

Analytical chromatographic column: SinoChrom ODS-BP, 250mm×4.6mm (5μm packing);

Mobile phase: A phase is water (100%), B phase is acetonitrile (100%);

Loading volume: 20μL;

Flow rate: 1mL/min;

Detection wavelength: 220nm;

Elution gradient: see Table 1.

Table 1. Elution gradients for detection of modification yields

Its mass spectrometry report and high performance liquid chromatography report are shown in Figure 1 and Figure 2. The results showed that the relative molecular mass of RS-17 was 2120.29 g/mol, and the purity of the synthesized RS-17 polypeptide was more than 90%.

Example 2 Analysis of MST intermolecular interaction between polypeptide RS-17 and CD47 protein

Microcalorimetry (MST) is a technique for analyzing biomolecules. Microscale thermophoresis is the directional motion of particles in microscopic temperature gradients. The relative change in motion along a temperature gradient caused by changes in the hydration layer due to changes in biomolecular structure/conformation can be used to determine affinity (K _d value).

In the examples of this part, the polypeptide RS-17 (RRYKQDGGWSHWSWPWSS-NH ₂ ) is mainly used as the object to study the interaction between it and CD47 extracellular segment recombinant proteins (Ala18-Glu141).

1.1 Experimental materials

Polypeptide RS-17 (RRYKQDGGWSHWSPWSS-NH ₂ ); human recombinant protein CD47 (Ala18～Glu141, Cloud-Clone, USA)

1.2 Experimental instruments

Monolith NT.115MST microcalorimeter (nanotemper, Germany); CapillariesMonolith NT.115 standard capillary (nanotemper, Germany); microcentrifuge (thermo, USA)

1.3 Main reagents

Sulfo CY5 MAL (Xi'an Ruixi Biotechnology Co., Ltd.); PBS buffer (pH=7.2, gibco, USA)

1.4 Experimental method

Preparation of tagged protein: put the recombinant protein CD47 in a buffer that does not contain primary amine groups (ammonium ion free, triisopropylacetamide Tris, glycine, ethanolamine, triethylamine, glutathione or imidazole), Adjust the final concentration of recombinant protein to 260 nmol/L. Dilute the corresponding labeling dye to a concentration of 2000 times the recombinant protein. Mix protein and labeling dye 1:1 and incubate at room temperature for 30 min in the dark. Then add the protein dye mixture to the B column of the MST microcalorimeter, open the bottom cover of the B column, put it into a 15ml centrifuge tube, and collect the eluate by gravity.

Preparation of unlabeled polypeptide: Adjust the concentration of the polypeptide to 20 times that of the labeled protein (5200nmol/L) and carry out gradient dilution into 16 parts (2600, 1300, 650, 325, 162.5, 81.25, 40.625, 20.3125, 10.15625, 5.078125, 2.5390625, 1.26953125, 0.634765625, 0.3173828125, 0.1586914063, 0.07934570313).

Preparation and loading: The labeled protein and the unlabeled polypeptide were mixed 1:1 in a 200 μL EP tube and incubated for 40 minutes. The final concentration of the labeled protein in each tube was 130 nmol/L.

MST analysis: Use a standard capillary to draw samples of each concentration, put them in the measuring tank in sequence, and close the front of the machine. Open the NT Control software, set the LED Power[%] to 40, click Start Cap Scan to scan the capillary, and scan whether each sample is kept neat. Then click Start MST Measurement to measure.

Data analysis: NT Analysis software was used to analyze, generate a fitting curve, and calculate the K _d value.

1.5 Experimental results

Thermophoretic amplitude showed dissociation between CD47-RS-17. As shown in Figure 3, the binding curve shows that with the increase of RS-17 concentration, the relative fluorescence of MST of CD47 protein changes, and there is a strong binding between CD47 and RS-17, which increases with the increase of RS-17 concentration . The best fitting curve was obtained by software analysis, and the K _d value of the binding of CD47 to RS-17 was 3.857±0.789nM.

Example 3

Western blot detected tumor cell lines with high CD47 expression and lymphocyte cell lines with high expression of SIRPα as positive cells, which were subsequently used to detect the blocking effect of peptides on CD47/SIRPα pathway.

1.1 Experimental materials

Human hepatoma cell HepG2: Shanghai Chunmai Biological Company; human epidermal squamous cell carcinoma SCC-13: Human mononuclear lymphoma cell THP-1: Shanghai Chunmai Biological Company; mouse macrophage lymphoma cell RAW264.7: cell culture Bottle of American Thermo Company; cell culture medium: American Gibco Company; Rabbit anti-human/mouse CD47 antibody: American BioXcell Company; Rabbit anti-human/mouse SIRPα antibody: Wanclass Biotechnology Co., Ltd.; Rabbit anti-human/mouse GAPDH antibody: Lianke Bio Co., Ltd. HRP Goatanti-Rabbit IgG: LinkBio Co., Ltd.

1.2 Experimental instruments

Inverted microscope (OLYMPUS company in Japan), small centrifuge (Thermo company in the United States), electronic balance (Sartorius company in Germany), pH meter (Shanghai Lei magnetic company), ultra-clean bench (Suzhou purification company).

1.3 Experimental method

The cell lines HepG2, SCC-13, RAW264.7 and other cell lines used in the experiment used high glucose DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), streptomycin (100mg/L), THP The -1 cell line adopts RMPI 1640 medium containing 10% fetal bovine serum (FBS), penicillin (100 kU/L), streptomycin (100 mg/L), and β-mercaptoethanol (0.05 mM). Incubate at 37°C in a 5% CO2 incubator until logarithmic growth phase. The cell concentration was adjusted to 2x105/ml, and 100 μl/well was seeded in a 96-well plate. Incubate for 24h at 37°C in an incubator containing 5% CO2.

The cultured cells are then used to prepare protein samples, the medium is discarded, and the cells are processed into cell suspension. The cell suspension was added to a 2ml centrifuge tube, centrifuged at 800rpm for 5min, and the supernatant was discarded. Add 1 ml of PBS to the centrifuge tube to resuspend the cells, centrifuge at 800 rpm for 5 min and discard the supernatant. Prepare cell lysate: Measure an appropriate amount of cell lysate and add PMSF to a final concentration of 1 mM. Mix well and place on ice for later use. The lysed cells were dropped into a centrifuge tube, the cells were mixed, placed on a shaker, and the cells were lysed in an ice bath for 30 min. After the lysis was completed, centrifuge at 12,000 rpm for 10 min, and take the supernatant as the protein solution. Pellet as cell debris.

After quantification with BCA, Western blot was used to detect the expression of CD47 and SIRPα in each cell. Wash the electrophoresis plate, clamp it, and check for leaks with water. Configure 8% SDS-PAGE. Take an appropriate amount of protein samples for electrophoresis, the concentration gel constant pressure 100v, the separation gel constant pressure 140v, when the target band runs to the middle of the separation gel, stop electrophoresis. Wet transfer method. The protein was transferred from the gel to a PVDF membrane with a pore size of 0.22 μm, and the constant current was 100 mA for 100 min. In order to distinguish the protein side, the upper right corner of the PVDF membrane can be cut off. After transfer, the PVDF membrane was placed in 5% nonfat dry milk to block for 2h. Incubate the primary antibody at a dilution of 1:1000, fix it on a rotary mixer, and incubate overnight at 4°C. The membrane was washed 3 times with TBST, 10 min each time. Incubate the secondary antibody at a dilution of 1:4000, fix it on a rotary mixer, and incubate at room temperature for 2h. The membrane was washed 3 times with TBST, 10 min each time. In the dark room, put the PVDF membrane into the luminescent box, with the protein side up, gently wipe off the remaining TBST on the surface with paper, and then add the luminescent solution dropwise, and react for 5 min. Cut a piece of X-ray film that is the same size as the film, and press the film after a strip appears on the PVDF film. The time of pressing is determined by the brightness of the strip. Remove the X film, rinse it in the developer solution until the bands appear clearly, put it in the stop solution for 1 min, and finally rinse it in the fixer solution until the background is transparent, and rinse the fixed image with running water.

1.4 Experimental results

The expressions of different cell surface receptors CD47 and SIRPα are shown in Figure 4. The cell lines from left to right are: SCC-13, HEPG2, THP-1, RAW264.7. It can be seen from the figure: THP-1 and RAW264.7 highly express SIRPα, and all four types of cells highly express CD47.

Example 4 Detection experiment of the specific binding of polypeptide RS-17 to the target at the cellular level

Flow cytometry is a method that excites a single unidirectional flow of particles by a laser beam, and detects the scattered light of the particles and the fluorescent markers carried by them, so as to complete the rapid detection and analysis of multiple physical properties of a single particle and cell sorting. technology. Widely used in scientific research and clinical medical testing, it is the most advanced cell quantitative analysis technology. The main target of a polypeptide with cancer-preventing and/or tumor-inhibiting activity involved in the present invention is CD47. In this study, the designed anti-CD47 polypeptide RS-17 (RRYKQDGGWSHWSWPWSS-NH ₂ ) was linked to a FITC fluorescent marker, and the cell The binding rate to the FITC-anti-CD47 polypeptide solution reflects the binding ability of the polypeptide to CD47 on the cell surface at the cellular level.

1.1 Experimental materials

Human hepatoma cell HepG2: Shanghai Chunmai Biological Company; human epidermal squamous cell carcinoma SCC-13: polypeptide RS-17: synthesized in our laboratory, with FITC fluorescent label at the N-terminus.

1.2 Experimental instruments

Flow cytometer (Beckmancoulter, USA), inverted microscope (OLYMPUS, Japan), small centrifuge (Thermo, USA), electronic balance (Sartorius, Germany), pH meter (Shanghai Leici Company), ultra-clean bench (Suzhou Purification) company).

1.3 Experimental method

The cultured SCC-13 and HepG2 cells were collected and washed twice with ice-cold PBS. Add 1 ml of 1% BSA solution, fix it on a rotary mixer, and mix at 4°C for 30 min. FITC-antiCD47 polypeptide solution with a final concentration of 20ug/ml was added in the dark, fixed on a rotary mixer, and mixed at 4°C for 1 h in the dark. After incubating the drug, centrifuge at 800 rpm for 5 min and discard the supernatant. Wash once with ice-cold PBS. The cell concentration was adjusted with PBS, and the sample concentration was adjusted to 1 x 106 cells/ml. Do 3 parallels for each cell, and prepare 0.5ml for each sample.

Use a flow cytometer to detect the binding of peptides to CD47, turn on the regulated power supply, transformer, flow cytometer host, computer and printer in turn from left to right. Open the fluid flow drawer and add purified water to the sheath fluid bucket until it reaches 2/3. The waste liquid was poured out, and 200 ml of sodium hypochlorite solution containing 10% available chlorine concentration was added. Adjust the hydraulic valve to the pressurize position to remove air bubbles between the fluid line and the filter. Remove the sample tube, perform the PRIME function twice, 1ml PBS, HINGRUN2min. Start measuring and analyze the sample. After the measurement is complete, move the sample holder to the left and vacuum 1ml of FACSClean. Then return the sample support frame to the normal position, and HING RUN for 5min. Change the FACS Clean to pure water, move the sample support rack to the left, extract 1 ml of vacuum, return the sample rack to the positive position, and HING RUN for 10 minutes. Press Standby, remove the sample tube, and perform the PRIME function twice. Finally, 1 ml of purified water was left in the flow test tube. Press Standby to make it work for 20min, after the fan cools the laser, turn off the flow cytometer. Exit the program and shut down the computer. Experimental results:

1.4 Experimental results

The binding of SCC-13 cells to FITC-conjugated anti-CD47 polypeptide is shown in Figure 5. In the figure, the red peak is the negative control, and the blue peak is the binding when the polypeptide concentration is 20ug/ml. The binding rate is 55.5%. The binding of HepG2 cells to FITC-conjugated anti-CD47 polypeptide is shown in Figure 6. In the figure, the red peak is the negative control, and the blue peak is the binding when the peptide concentration is 20ug/ml, and the binding rate is 71.2% .

Example 5 Qualitative detection experiment of specific binding of polypeptide to target at the cellular level—fluorescence imaging experiment

1.1 Experimental materials

Human hepatoma cell HepG2: Shanghai Chunmai Biological Company; Human Epidermal Squamous Carcinoma Cell SCC-13: Polypeptide RS-17: Synthesized in our laboratory, N-terminal carrying FITC fluorescent label; DiI (cell membrane red fluorescent probe): Biyuntian Biotechnology Technology Co., Ltd.

1.2 Experimental instruments

Fluorescence inverted microscope (OLYMPUS company in Japan), small centrifuge (Thermo company in the United States), electronic balance (Sartorius company in Germany), ultra-clean bench (Suzhou purification company).

1.3 Experimental method

The concentration of SCC-13/HepG2 cells was adjusted to 1×10 ⁵ cells/ml and plated on a 24-well plate. Wash twice with ice-cold PBS. 1 ml of 1% BSA solution was added and mixed at 4°C for 30 min. Add FITC-anti CD47 polypeptide solution with a final concentration of 20ug/ml in the dark, and mix for 1 h at 4°C in the dark. After incubating drugs, wash once with ice-cold PBS. Dil dye was added and washed twice with ice-cold PBS.

Fluorescence microscopy was used to detect the binding of the polypeptide to CD47, and the parameters were fixed, and the images were taken in the appropriate channels respectively, and the fluorescence images of different channels were superimposed to judge the binding of the polypeptide to CD47.

1.4 Experimental results

Under the polypeptide concentration of 20ug/ml, the results of HepG2 immunofluorescence showed that the polypeptide RS-17 could bind to HepG2 with high expression of CD47. The results of SCC-13 immunofluorescence at the polypeptide concentration of 20ug/ml, we can see from the figure that the polypeptide RS-17 can bind to SCC-13 that highly expresses CD47.

The anti-CD47 polypeptide was verified again by fluorescence microscopy to bind to tumor cells with high expression of CD47, and to bind on the cell membrane surface.

Example 6 Polypeptide functional verification—promoting macrophage phagocytosis

The anti-tumor effect of RS-17 polypeptide is mainly achieved by blocking the interaction between CD47-SIRPα. The most direct result of this blocking effect is to cause the target tumor cells to be more easily phagocytosed by macrophages. Therefore, the functional verification of the polypeptide RS-17 in this example was achieved through a phagocytosis experiment.

1.1 Experimental materials

Human hepatoma cell HepG2: Shanghai Chunmai Biological Company; human mononuclear lymphoma cancer cell THP-1: Shanghai Chunmai Biological Company; mouse macrophage lymphoma cell RAW264.7: CD47 antibody B6H12: American BioXcell Company; IgG1 counterpart Control antibody: American thermo company; CFSE cytoplasmic dye: American thermo company; PMA: Biyuntian Biotechnology Co., Ltd.; LPS: Wanlei Biotechnology Co., Ltd.; IFNγ: American proteintech company.

1.2 Experimental instruments

Fluorescence inverted microscope (OLYMPUS company in Japan), small centrifuge (Thermo company in the United States), electronic balance (Sartorius company in Germany), ultra-clean bench (Suzhou purification company).

1.3 Experimental method

THP-1 monocytes were induced to M0 macrophages by PMA at a final concentration of 100ng/ml for 48h. After they adhered stably, they were induced to M1-type macrophages for 24 hours by the final concentration of 20ng/ml IFNγ and 1μg/ml LPS. RAW264.7 mouse macrophage lymphoma cells were induced to M1 type macrophages for 24 h by IFNγ at a final concentration of 20 ng/ml and LPS at 1 μg/ml. Then, the two kinds of cells were plated on 12-well plates at 2×10 ⁴ cells/ml for use.

HepG2 cells were collected. Wash twice with ice-cold PBS. 1 ml of 1% BSA solution was added and mixed at 4°C for 30 min. Add anti-CD47 antibody or polypeptide solution with a final concentration of 20ug/ml in the dark, and mix for 1 h at 4°C in the dark. After incubating the drug, wash once with ice-cold PBS. Then add CFSE dye to incubate for 15 min, and wash twice with ice-cold PBS. Then adjust the concentration to 1×10 ⁵ cells/ml and spread them in a 12-well plate where macrophages had been plated before. The two cells were incubated together for 2 h, and then the HepG2 cells engulfed inside the macrophages were observed by fluorescence microscope (green fluorescence) , 200 macrophages were randomly selected to count the internal HepG2 cells, and the phagocytosis index (the number of cells/microspheres phagocytosed by every 100 macrophages) was counted.

1.4 Experimental results

Figure 7 shows the results of co-incubating CFSE dye-labeled HepG2 with THP-1 macrophages for 2 hours under a 10x objective and a peptide concentration of 20ug/ml. From the figure, we can see that the peptide RS-17 Or under the action of CD47 antibody B6H12, the phagocytosis efficiency of macrophages against tumor cells was greatly improved. Figure 8 shows the results of co-incubating CFSE dye-labeled HepG2 with RAW264.7 macrophages for 2 hours under a 10x objective and a peptide concentration of 20ug/ml. From the figure, we can see that the peptide RS-17 Or under the action of CD47 antibody B6H12, the phagocytosis efficiency of macrophages against tumor cells was greatly improved.

Figure 9 shows the statistical results of the phagocytosis index for two macrophage conditions, THP-1 and RAW264.7. It was further verified that the presence of RS-17 and CD47 antibody B6H12 enhanced the phagocytic efficiency of macrophages (P<0.001).

Embodiment 7 In vivo efficacy test—polypeptide RS-17 acute toxicity detection

1.1 Experimental materials

Balb/c mice: Nanjing Qinglongshan Laboratory Animal Co., Ltd.; polypeptide RS-17: synthesized in our laboratory; 0.6% sodium chloride injection: Luyao Group Co., Ltd.

1.2 Experimental method

Balb/c mice were divided into 5 groups with five mice in each group. Each group was injected with 100mg/kg, 75mg/kg, 50mg/kg, 25mg/kg and 0mg/kg of RS-17 polypeptide through tail vein respectively. Breed for 24 hours. The death of mice was counted.

1.3 Experimental results

The results showed that none of the five groups of mice died within 24 hours, and all groups of mice had no abnormality after one week, and the polypeptide RS-17 did not have any serious physiological toxicity.

Example 8 In vivo efficacy test—Tumor formation of tumor cells in experimental animals after treatment with polypeptide RS-17

The anti-tumor effect of RS-17 polypeptide is mainly achieved by blocking the interaction between CD47-SIRPα. The most direct result of this blocking effect is to cause the target tumor cells to be more easily phagocytosed by macrophages. Therefore, tumor cells blocked by RS-17 polypeptide or CD47 antibody should be more easily cleared by the immune system after inoculation into experimental animals.

1.1 Experimental materials

Balb/c nude mice: Changzhou Cavens Laboratory Animal Co., Ltd.; polypeptide RS-17: synthesized in our laboratory; CD47 antibody B6H12: American BioXcell; HepG2-GFP (green fluorescence) human hepatoma cells: Shanghai Chunmai Biotechnology Technology Co., Ltd.; 0.6% Sodium Chloride Injection: Luyao Group Co., Ltd.

1.2 Experimental method

HepG2-GFP cells were collected. Wash twice with ice-cold PBS. 1 ml of 1% BSA solution was added and mixed at 4°C for 30 min. Add anti-CD47 antibody or polypeptide solution with a final concentration of 20ug/ml in the dark, and mix for 1 h at 4°C in the dark. After the incubation of the drug, the cell concentration was adjusted to 1×10 ⁸ cells/ml and washed twice with ice-cold PBS.

Balb/c nude mice were divided into three groups (IgG1, B6H12, RS-17), 3 mice in each group, and 0.1 ml of the previously treated HepG2-GFP cells were injected into the left armpit respectively. , tumor volume, tumor volume is calculated according to the formula V=Π/6ab ² , a, b are tumor length and tumor width respectively. On the seventh day, in vivo imaging of small animals was performed to calculate the mean fluorescence intensity of GFP in mice.

1.3 Experimental results

The changes in body weight and tumor volume of mice within seven days are shown in Figure 10. From the figure, we can see that the degree of weight loss and tumor volume increase of mice in the IgG1 group are stronger than those of B6H12 (positive control) and RS. -17 group (P<0.001), while there was no significant difference between the positive control and RS-17.

The average fluorescence intensity of GFP in vivo of mice on the seventh day is shown in Figure 11. From the figure, we can see that the average fluorescence intensity of GFP in the IgG1 group is also much higher than that of the other two groups, as shown in Figure 11. As can be seen from the figure, the mean fluorescence intensity of GFP in the IgG1 group was also much higher than that in the other two groups (P<0.001). It is shown that tumor cells treated with RS-17 are difficult to form tumors in experimental animals, and its functional mechanism can also be established in vivo.

The average fluorescence intensity of GFP in vivo of mice on the seventh day is shown in Figure 11. From the figure, we can see that the average fluorescence intensity of GFP in the IgG1 group is also much higher than that in the other two groups (P<0.001). It is shown that tumor cells treated with RS-17 are difficult to form tumors in experimental animals, and its functional mechanism can also be established in vivo.

Example 9 In vivo efficacy test—evaluation of polypeptide RS-17 tumor suppressor efficacy

1.1 Experimental materials

Balb/c nude mice: Changzhou Cavens Laboratory Animal Co., Ltd.; polypeptide RS-17: synthesized in our laboratory; HepG2-GFP (green fluorescence) human hepatoma cells: Shanghai Chunmai Biotechnology Co., Ltd.; 0.6% chloride Sodium Injection: Luyao Group Co., Ltd.

1.2 Experimental method

HepG2-GFP cells were collected. After washing twice with ice-cold PBS, the cell concentration was adjusted to 1×10 ⁸ cells/ml for use.

Balb/c nude mice were divided into two groups (Control, RS-17), 4 mice in each group, and 0.1 ml of HepG2-GFP cells collected in the front were injected into the left armpit respectively, and waited for the tumor to grow to a certain extent (>0.1 cm ^{3 )} . ). The RS-17 group was injected with RS-17 polypeptide at a dose of 10 mg/kg once a day, and the control group was injected with the same volume of normal saline. The mice were injected continuously for 21 days, and the body weight and tumor volume of the mice ^were measured every day. On the 21st day, a small animal in vivo imaging was performed to calculate the average fluorescence intensity of GFP in mice. And all the data were counted to calculate the tumor inhibition rate. The tumor inhibition rate was calculated according to the following formula:

Tumor volume (TV)=0.52ab ² , a and b are tumor length and tumor width, respectively.

Relative tumor volume (RTV)=V _t /V ₀ , V ₀ is the tumor volume measured at the start of administration, and V _t is the tumor volume at each measurement.

Relative tumor proliferation rate (T/C (%)) = T _RTV /C _RTV × 100%, T _RTV is the RTV of the treatment group; C _RTV is the RTV of the negative control group.

1.3 Experimental results

The changes in body weight and tumor volume of mice within 21 days are shown in Figure 12. From the figure, we can see that the degree of weight loss and tumor volume increase of the mice in the Control group are stronger than those in the RS-17 group (P <0.001).

The changes in relative tumor volume and relative tumor proliferation rate of mice within 21 days are shown in Figure 13. From the figure, we can see that the relative tumor volume of the mice in the Control group increased more strongly than that in the RS-17 group (P< 0.001), the tumor growth rate in the Control group was much faster than that in the RS-17 group. However, the relative tumor proliferation rates of the Control group and the RS-17 group had been decreasing, indicating that the RS-17 polypeptide played a role in inhibiting tumor growth.

The average fluorescence intensity of GFP in vivo of mice on the 21st day is shown in Figure 14. From the figure, we can see that the average fluorescence intensity of GFP in the Control group is also higher than that in the RS-17 group (P<0.05). showed that tumor cell growth was inhibited by RS-17 treatment, and its functional mechanism was also established in vivo.

sequence listing

<110> China Pharmaceutical University

<120> A polypeptide RS-17 with anti-CD47 immune checkpoint antagonistic activity and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> PRT

<213> Human co-sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 1

Arg Arg Tyr Lys Gln Asp Gly Gly Trp Ser His Trp Ser Pro Trp Ser

1 5 10 15

Ser

Claims (4)

1. A polypeptide RS-17 having anti-CD47 immune checkpoint antagonistic activity, characterized in that its amino acid sequence is: RRYKQDGGWSHWSPWSS-NH₂。

2. Use of the polypeptide RS-17 of claim 1 having anti-CD47 immune checkpoint antagonist activity for the preparation of a medicament for the treatment of cancer; the cancer is epithelial tissue cancer, lymphoma, blastoma, sarcoma, leukemia, lung cancer, peritoneal cancer, hepatocarcinoma, gastric cancer, pancreatic cancer, gallbladder cancer, cervical cancer, ovarian cancer, bladder cancer, breast cancer, and colon cancer.

3. A pharmaceutical composition characterized by: the composition contains the polypeptide RS-17 of claim 1 and one or more pharmaceutically acceptable auxiliary materials.

4. The pharmaceutical composition of claim 3, wherein: the medicine composition is prepared into injection and dry powder injection.

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