Anti-tumor oligopeptide, pharmaceutical composition and application thereof

Abstract

The invention discloses an anti-tumor oligopeptide. The oligopeptide comprises an amino acid sequence as shown in Seq ID No: 1; the N end of the amino acid sequence as shown in Seq ID No: 1 is also connected with a cell-penetrating peptide; the oligopeptide inhibits tumor growth by inducing tumor cell differentiation; the tumor is glioma; the oligopeptide is an oligopeptide from an LGP2 source. The invention also discloses an anti-tumor pharmaceutical composition containing a PD-1 antibody and the short peptide. The invention also discloses an application of the oligopeptide or the antitumor drug composition in preparation of antitumor drugs. The invention provides a novel differentiation treatment approach for tumors, especially glioma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-tumor short peptide, a pharmaceutical composition and application thereof. Background Art

[0002] Differentiation therapy is an innovative treatment strategy that inhibits malignant proliferation by reactivating the differentiation potential of cancer cells and promoting their development to a mature state. This therapy is different from traditional cytotoxic therapy. It does not directly kill cancer cells, but reduces their tumorigenicity by changing the differentiation state of cancer cells. It has attracted much attention in recent years because of its fewer side effects, limited impact on normal cells and lasting efficacy. The earliest successful case came from the application of all-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APL). This therapy degraded the PML-RARα fusion protein, relieved the differentiation block of leukemia cells, and significantly improved the patient's prognosis. Currently, with the development of molecular biology technology, differentiation therapy is gradually revealing its potential new targets in the treatment of malignant tumors and is gradually being used in solid tumors such as neuroblastoma and hepatocellular carcinoma. However, the therapeutic effect on solid tumors and the specific mechanism of inducing differentiation are still unclear.

[0003] The RIG-I-like receptor family (RLRs) includes retinoic acid-inducible gene-I (RIG-I), melanoma differentiation-associated receptor 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). They can interact with exogenous RNA in cells to cause conformational changes. After MDA5 and RIG-I sense non-self RNA, their CARD domains are exposed. The exposed CARD interacts with the CARD domain of mitochondrial antiviral signaling protein (MAVS) to promote the activation of transcriptional regulators including IRF-3 and NFκB, leading to the transcription of type I interferon (IFN) and IFN-inducible genes (ISGs), and participates in the immune response to viral infection. IFN and ISGs can not only affect the replication of viruses, but also have a direct tumor-killing effect. Although LGP2 can bind to RNA and binds to RNA more firmly than MDA5 and RIG-I, it lacks the CARD domain or other signal transduction domains and cannot transmit signals, resulting in the inability to directly interact with the adaptor protein MAVS to induce the production of IFN and inflammatory factors. Currently, the function of LGP2 in immune response is still controversial. A large number of studies have shown that LGP2 has a promoting effect in MDA5 antiviral signaling. LGP2 is mainly involved in innate immunity and antiviral immunity, and its role in tumors has not been reported. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an anti-tumor short peptide, a pharmaceutical composition and application thereof.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] In one aspect, the present invention provides an anti-tumor short peptide, wherein the short peptide comprises an amino acid sequence as shown in Seq ID No: 1.

[0007] As a further improvement of the present invention, the N-terminus of the amino acid sequence shown in Seq ID No: 1 is also connected to a cell-penetrating peptide.

[0008] Furthermore, the amino acid sequence of the cell-penetrating peptide is shown in Seq ID No: 2.

[0009] Furthermore, the short peptide inhibits tumor growth by inducing tumor cell differentiation.

[0010] Furthermore, the tumor is a glioma.

[0011] Furthermore, the short peptide is a short peptide derived from LGP2.

[0012] On the other hand, the present invention also provides an anti-tumor pharmaceutical composition comprising a PD-1 antibody and the above-mentioned short peptide.

[0013] In another aspect, the present invention also provides a use of the above-mentioned short peptide in the preparation of anti-tumor drugs.

[0014] In another aspect, the present invention also provides a use of the above anti-tumor pharmaceutical composition in the preparation of anti-tumor drugs.

[0015] The present invention studies the anti-tumor activity of the above-mentioned short peptide (H1), detects its effect on the survival of glioma cell lines U87MG and T98G, detects its effect on the growth and clone formation ability of glioma organoids and the effect of inducing the expression of differentiation markers. Subsequently, in an experiment in which gliomas were implanted intracranial in BALB / c nude mice, the growth of the tumor was observed. The results show that H1 can be used as a short peptide that inhibits tumor growth and induces differentiation. Other experiments have shown that the H1 short peptide can enhance the effect of PD-1 antibody therapy and can be used in combination for anti-tumor. The present invention provides a new differentiation therapy approach for tumors, especially gliomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The above is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0017] Figure 1 This is a graph showing that H1 short peptide inhibits the growth of glioma cells and induces the expression of differentiation markers, where:

[0018] A is a graph showing the effect of H1 on the survival of U87MG and T98G cells; B and C are graphs showing the effect of H1 on the clone formation of U87MG and T98G cells; Figure D is a graph showing the effect of H1 on the growth of glioma organoids; Figures E and F are graphs showing the effect of H1 on the levels of organoid differentiation marker proteins;

[0019] Figure 2 The H1 short peptide significantly inhibits the growth of glioma in tumor-bearing mice and induces glioma cell differentiation; among them:

[0020] A is a schematic diagram of the U87MG-Luc tumor transplant; B and C are bioluminescent images of mouse orthotopic gliomas on the 14th day after tumor implantation (n=3) and the corresponding fluorescence numerical statistical analysis graphs; D is a graph showing the size of the orthotopic gliomas (within the blue dotted box) by HE staining; E is a graph of the protein expression levels of differentiation markers TUBB3 and GFAP in four groups (H1, Hnc, PBS, TMZ) of mouse brain gliomas after H1 treatment detected by immunohistochemistry; F is a graph of mouse survival curve analysis.

[0021] Figure 3 This is a diagram showing that H1 short peptide significantly enhances the effect of PD-1 antibody treatment, where:

[0022] A is a schematic diagram of the Gl261-Luc tumor transplant; B and C are bioluminescent images of mouse orthotopic gliomas on the 14th day after tumor implantation (n=3) and the corresponding fluorescence numerical statistical analysis graphs; D is a mouse survival curve analysis graph; E is a graph showing the size of the orthotopic glioma (within the blue dotted box) by HE staining; FH are immunohistofluorescence methods for detecting the levels of CD8 and TNFa in six groups of mouse brain gliomas (PBS, Hnc, H1, aPD-1, aPD-1+Hnc, aPD-1+H1) after treatment with H1 and PD-1 antibodies, as well as fluorescence numerical statistical analysis graphs of CD8 and TNFa. DETAILED DESCRIPTION

[0023] The present invention finds that LGP2 interacts with MCCC1 (methylcrotonyl-CoA carboxylase 1), upregulates the protein levels of MCCC1 and MAVS and promotes the interaction between MCCC1 and MAVS, thereby activating the production of downstream type I IFN and interferon-stimulated genes, and inhibiting glioma growth.

[0024] The present invention found through experiments that LGP2 can interact with MCCA, thereby inhibiting the enzymatic activity of MCC. Finally, ECHS1 was used to mediate the upregulation of intracellular crotonylation modification levels. Histone H3K18cr crotonylation modification enhanced the transcription level of PPARGC1A. PGC1a can affect mitochondrial fission by promoting DRP1 phosphorylation, producing an anti-Warburg effect, and ultimately driving glioma cell differentiation.

[0025] The H1 short peptide derived from LGP2 can mimic the function of LGP2. On the one hand, it interacts with MCCA and MCCB to inhibit the enzyme activity of MCC to induce differentiation. On the other hand, it stabilizes the protein of MCCA, thereby activating MAVS and its downstream pathways.

[0026] The H1 short peptide derived from LGP2 has the function of inhibiting glioma growth and inducing glioma cell differentiation in glioma. The following is an experimental explanation:

[0027] 1. Experimental methods:

[0028] (1) Cell culture

[0029] Cell lines U87MG, T98G and glioma organoids were cultured in Dulbecco's modified Eagle's medium (DMEM; Meilunbio: MA0212). The components of the medium included 10% fetal bovine serum (FBS) and antibiotics (penicillin (100U / ml) / streptomycin (0.1mg / ml)). Cell culture conditions were 5% CO2 and 37°C, and the medium was changed once a day.

[0030] (2) The H1 short peptide derived from LGP2 was synthesized by Nanjing Leon Biotechnology Co., Ltd., and Hnc was used as a negative control. The amino acid sequences of H1 and Hnc and the N-terminal transmembrane peptide (TAT) sequence are as follows:

[0031] H1:YGRKKRRQRRR-KDTVYNVIMSQYLELKL(Seq ID No: 2-Seq ID No: 1)

[0032] Hnc:YGRKKRRQRRR-KTRAAAYVAKRHLET(Seq ID No: 2-Seq ID No: 3)

[0033] It should be noted that the H1 and Hnc used in the following experiments contain transmembrane peptide (TAT)

[0034] However, the cell-penetrating peptide YGRKKRRQRRR (Seq ID No: 2) is only used to carry the corresponding sequence (Seq ID No: 1: KDTVYNVIMSQYLELKL) into the cell, and the sequence that actually plays an anti-tumor role should be the sequence it carries. In addition, in addition to the aforementioned cell-penetrating peptides, other types of cell-penetrating peptides can also be used. This embodiment only takes the cell-penetrating peptide (Seq ID No: 2) as an example for explanation.

[0035] (3) Western blot

[0036] In order to detect the induced expression of differentiation marker proteins by H1 short peptide, Western blot experiments were used for protein verification. Western and IP cell lysis buffer (Biyuntian, product number: P0013) was used to lyse glioma organoids and collect proteins. 20μg of total protein was taken from each group for protein electrophoresis. After transfer, the membrane was incubated with 5% skim milk at 22°C for 2 hours, and then incubated with the primary antibody at 4°C overnight. The main antibodies and their dilutions are as follows:

[0037] Beta-Actin Mouse mAb(Proteintech#23660-1-AP):Dilution-1:5000;

[0038] GFAP Mouse mAb(Proteintech#60190-1-Ig):Dilution-1:5000;

[0039] MAP2Rabbit mAb(Proteintech#17490-1-AP):Dilution-1:5000;

[0040] TUBB3Mouse mAb(Proteintech#66375-1-Ig;):Dilution-1:5000;

[0041] Subsequently, anti-rabbit secondary antibody (HRP-linked antibody (#7074, CST, USA): 1:5000) and anti-mouse secondary antibody (HRP-linked antibody (#7076, CST, USA): 1:5000) were used at room temperature for 1 hour. Protein bands were visualized using a chemiluminescent ECL kit (Tanon, Shanghai, China).

[0042] (4) CCK-8 experiment

[0043] Take U87MG and T98G cells in the logarithmic growth phase, centrifuge at 300g for 5 minutes, and collect the cell pellet. Resuspend the cells in fresh culture medium, dilute 100 times, and count the cells. Set up 5 experimental groups, 0μM, 1μM, 5μM, 10μM, 15μM, 5 replicates in each group, and set the concentration of 1x10 3 At a density of 100 cells / well, 100 μL of cell suspension was added to a 96-well plate. The corresponding concentration of H1 short peptide was added to the experimental group, and the Hnc short peptide was used as a control. After culturing for 72 hours at 37°C 5% CO2, 10 μL of CCK8 solution (yeasen, #40203ES76) was added to each well. The 96-well plate was placed in a cell culture incubator and cultured for another 2 hours. The absorbance value (OD value) at a wavelength of 450 nm was detected using an ELISA reader. The cell activity was obtained by calculating the formula OD experimental group / OD0 μM.

[0044] (5) Plate cloning experiment

[0045] 5x10 cells were seeded in a six-well plate 2 For U87MG or T98G cells, add 2mL of DMEM medium, shake gently back and forth and left and right to ensure that the cells are evenly distributed, and culture in a 37°C incubator. Replace with fresh medium every 3 days. After about 14 days of culture, discard the medium and wash once with PBS to remove excess medium components. Add 3mL of 4% paraformaldehyde (Suzhou Xinsaimei Biological) and fix the cells for 20 minutes at room temperature in the dark. Wash twice with PBS, then add 3mL of 0.1% crystal violet dye (Biyuntian C0121-100ml) and continue staining at room temperature in the dark for 20 minutes. Finally, wash twice more with PBS, dry the culture dish, and use a microscope to capture images of the clones.

[0046] (6) Immunofluorescence experiment

[0047] Carefully place the 12-well plate-specific slide in the 12-well plate and spread the glioma organoids in the wells. Wash the cells 3 times with PBS, making sure to absorb the residual liquid each time. Subsequently, add 4% paraformaldehyde to fix the cells at room temperature for 15 minutes, and then wash them again with PBS 3 times. Use 0.1% TritonX-100 to permeabilize the cells for 10 minutes. After permeabilization, wash twice with PBS. Then use 5% BSA (0.5g dissolved in 10mL PBS) to block for 1 hour at room temperature. Discard the BSA solution and add a 1:500 diluted primary antibody (150μL), including TUBB3 antibody and GFAP antibody. And maintain humidity and incubate overnight on a shaker at 4℃. After incubation, wash the membrane 4 times with PBS for 5 minutes each time. Add a fluorescent-labeled secondary antibody diluted 1:500 and incubate in the dark for 1 hour. After incubation, wash with PBS 4 times. Use DAPI to incubate in the dark for 5 minutes and then wash 4 times with PBS. Place the coverslip upside down on a glass slide with mounting medium and seal the edges with nail polish. Observe the fluorescence signal under a microscope.

[0048] (7) BALB / c nude mouse intracranial tumor experiment and H1 short peptide treatment

[0049] Six-week-old female BALB / c nude mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd. The mice were anesthetized and placed in a stereotaxic head frame. The entry point of a 1 ml syringe (Hamilton, Switzerland) was drilled into the coronal suture of each mouse, 2 mm to the right of the midline. The nude mice were divided into four groups.

[0050] 5×10 5U87MG-Luc cell suspension was injected into 10 μL buffer and injected for 5 minutes at a distance of 3 mm from the brain surface. After another 5 minutes, the syringe was removed and the injection point was sealed with bone wax. Starting from the 7th day, each mouse was stereotaxically injected with 10 μL H1 (2 mg / kg / 3d) or Hnc at the same site. Intraperitoneal injection of TMZ (25 mg / kg / 3d) or an equal amount of solvent PBS was used as a control. After 14 days, each mouse was intraperitoneally injected with 15 mg / ml D-luciferin by 100 μL, and in vivo images were obtained using the IVIS bioluminescence imaging system. The survival of the mice was recorded.

[0051] (8) HE staining and immunohistochemistry

[0052] Tumor tissues were fixed and embedded in paraffin. 4 μm thick tissue sections were taken and stained with hematoxylin-eosin (HE) for morphological observation. After dewaxing and antigen retrieval, other similarly prepared slides were incubated with primary antibodies overnight at 4°C, including anti-TUBB3 (1:500) or anti-GFAP (1:500). After thorough washing, they were incubated with secondary antibodies for 1 h, then dehydrated and sealed with neutral resin. Images were collected using Leica Aperio AT2 and Leica DM IRB instruments.

[0053] 2. Experimental results:

[0054] (1) LGP2-derived H1 short peptide can inhibit glioma growth and induce glioma cell differentiation.

[0055] Figure 1 The H1 short peptide inhibits the growth of glioma cells and induces the expression of differentiation markers. Figure 1 As shown, where:

[0056] A is the effect of H1 on the survival of U87MG and T98G cells. After adding 0μM, 1μM, 5μM, 10μM and 15μM H1 or Hnc short peptide to human glioma U87MG cells and T98G cells, CCK-8 was used to detect cell survival. The results showed that H1 can significantly inhibit cell activity.

[0057] B and C are the effects of H1 on the clone formation of U87MG and T98G cells. After adding 5μM H1 or Hnc for 14 days to human glioma U87MG cells and T98G cells, the clone formation ability of cells was detected by plate cloning experiment. The results showed that over-exposure to H1 can significantly inhibit the clone formation ability of cells.

[0058] D is the effect of H1 on the growth of glioma organoids. After 0d and 14d of treatment with 5μM H1 or Hnc, images were taken using a phase contrast microscope, and the area of ​​the organoids was quantitatively analyzed. The results showed that overexpression of H1 significantly inhibited the growth ability of organoids.

[0059] E, F are the effects of H1 on the protein levels of organoid differentiation markers. After 21 days of treatment with 5μM H1 or Hnc in glioma organoids, the protein expression levels of TUBB3 and GFAP in organoids were detected by Western blotting and immunofluorescence experiments. The results showed that H1 can induce the expression of glioma cell differentiation markers. "NS", not significant, *p<0.05, **p<0.01, ***p<0.001.

[0060] (2) H1 short peptide can significantly inhibit tumor growth.

[0061] Figure 2 The H1 short peptide significantly inhibited the growth of glioma in tumor-bearing mice and induced glioma cell differentiation. Figure 2 As shown, where:

[0062] A is a schematic diagram of U87MG-Luc tumor transplantation. 6 The glioma U87MG-Luc cells were orthotopically injected into the skull of BALB / c nude mice to construct an orthotopic mouse glioma transplant model. Seven days after transplantation, the tumor growth was monitored using the IVIS bioluminescence imaging system. H1 or Hnc short peptides were stereotactically injected into the tumor at a dose of 2 mg / kg / 3d per mouse, and TMZ (25 mg / kg / 3d) or PBS was intraperitoneally injected as a control. Tumor growth was monitored using the IVIS bioluminescence imaging system.

[0063] Figures B and C are bioluminescent images of mouse orthotopic glioma on day 14 after tumor implantation (n=3) and corresponding fluorescence numerical statistical analysis graphs. The results show that H1 short peptide can significantly inhibit the growth of glioma.

[0064] D is a HE staining showing the size of the in situ glioma (in the blue dotted box). After the experiment reached the end point, the mice were killed, and the coronal sections of the brain tissue were taken out and HE staining was performed to show the size of the glioma after treatment with H1, Hnc, PBS and TMZ. The results showed that compared with the control group, the tumors of intracranial gliomas in mice treated with Hnc and H1 were smaller, which was equivalent to the treatment effect of TMZ.

[0065] E is the immunohistochemical method to detect the protein expression levels of differentiation markers TUBB3 and GFAP in four groups (H1, Hnc, PBS, TMZ) of mouse brain glioma after H1 treatment. The results showed that compared with the control group Hnc and TMZ, H1 treatment could significantly induce glioma cell differentiation.

[0066] F is the mouse survival curve analysis diagram. The survival period of mice treated with H1, Hnc, PBS and TMZ. The results showed that compared with the control group Hnc, the survival period of mice treated with H1 was significantly prolonged, which was equivalent to the effect of TMZ treatment. "NS", not significant, *p<0.05, **p<0.01, ***p<0.001.

[0067] (3) H1 short peptide can enhance the effect of PD-1 antibody therapy.

[0068] Figure 3 This is a picture showing that the H1 short peptide significantly enhances the effect of PD-1 antibody treatment. Figure 3 As shown, where:

[0069] A is a schematic diagram of Gl261-Luc tumor transplantation. 6 The glioma GL261-Luc cells were orthotopically injected into the skull of C57 mice to construct an orthotopic mouse glioma transplant model. Seven days after transplantation, the tumor growth was monitored using the IVIS bioluminescence imaging system. H1 or Hnc short peptides were stereotactically injected into the tumor at a dose of 2 mg / kg / 3d per mouse, and PD-1 antibodies (10 mg / kg / 3d) or PBS were intraperitoneally injected as controls. Tumor growth was monitored using the IVIS bioluminescence imaging system.

[0070] B and C are bioluminescent images of mouse orthotopic glioma on the 14th day after tumor implantation (n=3) and the corresponding fluorescence numerical statistical analysis graphs. The results show that H1 short peptide can significantly inhibit the growth of glioma and H1 short peptide can significantly enhance the therapeutic effect of PD-1 antibody.

[0071] D is the mouse survival curve analysis chart. The survival period of mice treated with H1, Hnc, PBS and PD-1 antibody. The results showed that compared with the control group, the survival period of mice treated with PD-1 and H1 was significantly prolonged, and the combination of PD-1 and H1 was more effective than single-drug treatment.

[0072] E is a picture of HE staining showing the size of the in situ glioma (in the blue dotted box). After the experiment reached the end point, the mice were killed, and the coronal sections of the brain tissue were taken out and HE staining was performed to show the size of the glioma after treatment with H1, Hnc, PBS and PD-1. The results showed that compared with the control group, the tumors of intracranial gliomas in mice treated with PD-1 antibody and H1 were smaller, and the combination of PD-1 and H1 was more effective than single-drug treatment.

[0073] FH is an immunohistofluorescence method to detect the levels of CD8 and TNFa in six groups of mouse brain gliomas (PBS, Hnc, H1, aPD-1, aPD-1+Hnc, aPD-1+H1) after treatment with H1 and PD-1 antibodies, as well as the fluorescence numerical statistical analysis of CD8 and TNFa. The results showed that compared with the control group, PD-1 antibody and H1 treatment could significantly increase the levels of CD8 and TNFa in the tumor microenvironment. The combination of H1 and PD-1 antibodies can further increase the levels of CD8 and TNFa in the tumor microenvironment compared with single-drug treatment.

[0074] "NS", not significant, *p<0.05, **p<0.01, ***p<0.001.

[0075] In summary, the present invention studies the anti-tumor activity of the H1 short peptide, detects its effect on the survival of glioma cell lines U87MG and T98G, and detects its effect on the growth and clone formation ability of glioma organoids and the effect of inducing the expression of differentiation markers. Subsequently, in an experiment in which gliomas were implanted intracranial in BALB / c nude mice, the growth of the tumor was observed. The results show that H1 can be used as a short peptide that inhibits glioma growth and induces differentiation. Other experiments have shown that the H1 short peptide can enhance the effect of PD-1 antibody therapy and can be used in combination for anti-tumor.

[0076] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art may make some simple modifications, equivalent changes or modifications using the technical contents disclosed above, which all fall within the protection scope of the present invention.

Claims

1. An anti-tumor short peptide, characterized in that: The short peptide comprises the amino acid sequence shown in Seq ID No:

1.

2. The anti-tumor short peptide according to claim 1, characterized in that: The N-terminus of the amino acid sequence shown in Seq ID No: 1 is also connected to a cell-penetrating peptide.

3. The anti-tumor short peptide according to claim 2, characterized in that: The amino acid sequence of the cell-penetrating peptide is shown in SeqID No:

2.

4. The anti-tumor short peptide according to any one of claims 1 to 3, characterized in that: The short peptide inhibits tumor growth by inducing tumor cell differentiation.

5. The anti-tumor short peptide according to any one of claims 1 to 3, characterized in that: The tumor is a glioma.

6. The anti-tumor short peptide according to any one of claims 1 to 3, characterized in that: The short peptide is a short peptide derived from LGP2.

7. An anti-tumor pharmaceutical composition, characterized in that: Comprising a PD-1 antibody and the short peptide according to any one of claims 1 to 6.

8. Use of the short peptide according to any one of claims 1 to 6 in the preparation of anti-tumor drugs.

9. Use of the antitumor pharmaceutical composition according to claim 7 in the preparation of antitumor drugs.

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