CN106279399B

CN106279399B - Chemokine modified peptide

Info

Publication number: CN106279399B
Application number: CN201510301013.7A
Authority: CN
Inventors: 程家维; 郑锡聪; 游辉元
Original assignee: Beijing Rui Rui Biological Medicine Technology Development Co ltd
Current assignee: Beijing Rui Rui biological medicine technology development Co., Ltd.
Priority date: 2015-06-03
Filing date: 2015-06-03
Publication date: 2021-01-12
Anticipated expiration: 2035-06-03
Also published as: CN106279399A

Abstract

A modified chemokine peptide comprising an amino acid sequence having: (a) a N' -glutamic acid (E) -leucine (L) -arginine (R) signature sequence located at the N-terminus of the chemokine peptide; (b) n' -proline (P) -alanine (A) -serine (S) -glutamine (Q) -phenylalanine (F) -Cys³The 3 rd cysteine (Cys) of the chemokine peptide from the N-terminus³) (ii) a And (c) a modification position, wherein the modification position is located at the 17 th amino acid counted from the N end of the chemokine peptide. In addition, the modified chemokine peptides of the invention can be used to treat cancer, inhibit tumor growth.

Description

Chemokine modified peptide

Technical Field

The present invention relates to a modified chemokine useful as a therapeutic agent, and more particularly, to a modified chemokine useful for treating cancer and inhibiting tumor growth.

Background

Chemokines (also known as chemokines) are a group of small molecules (8-14kd) that can be induced, secreted and structurally similar. The chemokines can be divided into four subgroups CXC, CC, CX3C and C; if classified by function, chemokines can be classified into constant chemokines (Homeostasis chemokines) and Inflammatory chemokines (Inflammatory chemokines).

Chemokines typically have three β -sheets (β -sheets) in their structure, with an α -helix (α -helix) at the C-terminus and 4 cystines (cysteines) retained at the N-terminus. The first two cystine sequences contained at the N-terminus can be divided into four groups: CXC, CC, C, CX3C, and CC and CXC chemokines are the major. The reaction occurs after the binding of the cell chemokines to the chemokine receptors, which have 7G protein binding receptors spanning the cell membrane and are named CXCR, CCR, CXR, CX, depending on the type of ligand (ligand) to which they bind₃CR, if any, is further arranged in order of number, such as CXCR1, CXCR2, CCR4, etc. However, not only one chemokine receptor is expressed on target cells, but the inflammatory cell infiltrates bind to the target and the chemokine receptors in a non-one-to-one manner, and some chemokine receptors are expressed on different cells with some stimulation.

For example, ELR-CXC chemokines containing the characteristic sequence of glutamic acid (E) -leucine (L) -arginine (R) (ELR characteristic sequence) which is a protein having an amino acid sequence characteristic of ELR-CXC at the N-terminus of the protein, X may be a polar amino acid with or without an electrical property, or X is not present, and they regulate oncogene growth, IL-8, second type of neutrophil activating peptide (NAP-2), whose receptors are CXCR1, CXCR2, and whose primary acting cells are neutrophils, which promote accumulation and activation of neutrophils, are important and play a major role in the development of such ELR-CXC chemokines and a wide range of acute and chronic inflammatory disorders, and these inflammatory reactions include psoriasis and rheumatoid arthritis.

ELR-CXC chemokines are also more involved in angiogenesis associated with tumor development, the mechanism of induction being activation through the binding of such chemokines, particularly IL-8, to CXCR1 and CXCR2 on vascular Endothelial Cells (ECs). There are many different types of tumors that have been demonstrated to produce ELR-CXC chemokines, and tumors that express such chemokines have been thought to be associated with poor prognosis development of tumors.

By antagonizing the binding of CXCR1 or CXCR2 to ELR-CXC chemokines, a feasible strategy to inhibit aberrant signaling triggered by activation of CXCR1 or/and CXCR2 receptors, and thus treat related diseases caused by activation of cells bearing both receptors, and scientists are working on finding and preparing receptor protein analogs that inhibit CXC chemokines.

The present invention has been made in view of the above-mentioned problems, and it is an object of the present invention to provide a novel and effective method for overcoming the above-mentioned problems.

Disclosure of Invention

Cancer is one of the major causes of death in developed countries. Although there are advances in cancer diagnosis and treatment, and surgery and radiotherapy can cure cancer if it can be found early, most drugs have not been effective or have significant side effects. Therefore, there is a need in the medical field for a method and composition for treating or preventing cancer.

In view of the above, the present invention develops a modified chemokine peptide for inhibiting tumor growth and treating cancer.

The present invention provides a modified chemokine peptide comprising an amino acid sequence having an N-terminus (N') with: (a) a characteristic sequence of N' -glutamic acid (E) -leucine (L) -arginine (R), which is located at the N-terminus of said chemokine peptide; (b) n' -proline (P) -alanine (A) -serine (S) -glutamine (Q) -phenylalanine (F) -Cys³The 3 rd cystine (Cys) from the N-terminal of the chemokine peptide³) (ii) a Wherein the chemokine peptide contains the characteristic sequences of (a) and (b) and has a modification position, and the modification position is located at 17 th, 12 th and 13 th amino acids counted from the N end on the chemokine peptide.

In one embodiment, the chemokine peptide has the 17 th amino acid replaced by phenylalanine (F) to leucine (L).

In one embodiment, the unmodified precursor of the modified chemokine peptide is derived from a source chemokine peptide, wherein 0-2 amino acids can exist between the 1 st cystine and the 2 nd cystine at the N-terminal of the source chemokine peptide, and when the amino acids are 1-2, the amino acids are polar amino acids with or without electrical property.

In one embodiment, the source chemokine peptide is one selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9, and combinations thereof.

In one embodiment, the modified chemokine peptide of the invention is selected from one of the group consisting of SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, and combinations thereof.

The invention also provides a pharmaceutical composition comprising the modified chemokine peptide of the invention and a pharmaceutically acceptable excipient.

In one embodiment, the use of the modified chemokine peptide, wherein the use is for treating cancer or inhibiting tumor growth.

The invention also provides a pharmaceutical composition for treating cancer and inhibiting tumor, which comprises the modified chemokine peptide and a pharmaceutically acceptable excipient.

In one embodiment, the cancer includes prostate cancer, breast cancer, uterine cancer, blood cancer, ovarian cancer, endometrial cancer, cervical cancer, colorectal cancer, testicular cancer, lymphatic cancer, rhabdomyosarcoma, neuroblastoma, pancreatic cancer, lung cancer, brain tumor, skin cancer, stomach cancer, oral cancer, liver cancer, laryngeal cancer, biliary cancer, thyroid cancer, liver cancer, kidney cancer, nasopharyngeal cancer, and the like.

Drawings

FIG. 1 is an alignment chart of the amino acid sequences of the source chemokine peptide sequences (SEQ ID NO:1-SEQ ID NO:9) of the ELR-CXC chemokines of the present invention, which also have high affinity for CXCR1 or/and CXCR 2.

FIG. 2 is an alignment chart of amino acid sequences of the modified chemokine peptides of the invention (SEQ ID NOS: 10-12).

Figure 3 shows the number of cells migrating with the chemokine CXCL 8.

FIG. 4 shows the number of cells migrating with the chemical hormone stimulation in the black part, the chemical hormone stimulation and CXCL8-IP10 in the gray part, and the chemical hormone stimulation and IL8-17LIP10 in the white part, respectively.

FIG. 5 shows the tumor volumes of the experimental group (IL8-17LIP10) and the control group (saline).

FIG. 6 shows the tumor weights of experimental group (IL8-17LIP10) and control group (saline)

FIG. 7 shows the microvascular density of experimental group (IL8-17LIP10) versus control group (saline).

FIG. 8 is a graph showing the observation of the tumor volume after injection of CXCL8-IP10 and physiological saline, wherein:

group A: CXCL8-IP 10500 ug/kg with 4 injections per worship, group B: CXCL8-IP 10500 ug/kg 2 injections per worship, group C: CXCL8-IP 10250 ug/kg 2 injections per worship, group D: 100 μ l of saline was injected subcutaneously every day and the tumor volume was observed.

FIG. 9 shows sacrificed lung tumor tissue from mice, wherein A is the lungs removed after sacrifice from mice injected 4 times per worship with CXCL8-IP 10500 ug/kg on day 24; b is the lung of the rat injected with normal saline, wherein the white tumor site indicated by the arrow is the focus of metastasis to the lung.

Figure 10 shows the trend of expressing CXCR1 or CXCL8 in tumor cells.

FIG. 11 shows the effect of antagonists of the invention on chemotactic response.

Detailed Description

The present invention provides a novel chemokine peptide. The novel chemokine peptides of the invention are derived from a substrate belonging to the ELR-CXC chemokine or having a high affinity for CXCR1 or CXCR2, for example CXCL1(SEQ ID NO:3), CXCL2(SEQ ID NO:4), CXCL3(SEQ ID NO:5), CXCL5(SEQ ID NO:6), CXCL6(SEQ ID NO:7), CXCL7(SEQ ID NO:8), CXCL8(SEQ ID NO:2), hG31P (SEQ ID NO: 1). The invention is based on the modification of this type of chemokine by inserting, essentially at its 30s-loop, a PASQF signature sequence (see FIG. 1), which is originally present in the chemokine CXCL10, and the CXCL10 is a non-ELR-CXC chemokine.

The modified chemokines of the invention include: (a) a characteristic sequence of N' -glutamic acid (E) -leucine (L) -arginine (R), which is located at the N-terminus of the chemokine peptide; and (b) N' -proline (P) -alanine (A) -serine (S) -glutamine (Q) -phenylalanine (F) -Cys³The characteristic sequence of (1) is the 3 rd cystine (Cys) from the N-terminal of the chemokine peptide³). In addition, it should be noted that the chemokine peptide of the present invention further has a mutation position (i.e., a modification position) which is located at the 17 th amino acid counted from the N-terminus of the chemokine peptide.

In one embodiment, the 17 th amino acid of the chemokine peptide of the invention is originally phenylalanine (F) and is mutated or substituted into other amino acids other than phenylalanine. In another embodiment, the amino acid at position 17 of the chemokine peptide of the invention can be alanine (a), cysteine (C), selenocysteine (U), aspartic acid (D), asparagine (N), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), leucine (L), isoleucine (I), lysine (K), pyrrolysine (O), methionine (M), proline (P), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y), preferably leucine (L). Compared with the unsubstituted chemokine polypeptide, the chemokine polypeptide with the amino acid at the position 17 after being replaced has higher affinity and can effectively inhibit the growth of cancer cells.

In a specific embodiment of the invention, the modified chemokine peptides of the invention include, but are not limited to, SEQ ID NO 10, SEQ ID NO 11 and/or SEQ ID NO 12 (see FIG. 2).

Taking SEQ ID NO. 10 as an example, SEQ ID NO. 10 corresponds to ELR-CX except for having the N-terminal sequence of ELR-CQC_nC, SEQ ID NO:10 also has an oligopeptide sequence of Pro-Ala-Ser-Gln-Phe (proline-alanine-serine-glutamine-phenylalanine, PASQF) from the N-terminus, which PASQF is a modified sequence and is inserted upstream of the 3 rd Cysteine (Cysteine, C) from the N-terminus (10192), and which 3 rd Cysteine is immediately after the phenylalanine (F) of the PASQF oligopeptide sequence. More importantly, the amino acid at position 17 is not phenylalanine, but leucine.

As can be seen from the sequence listing provided by the present invention (see FIG. 2), the ELR-CXC chemokine analogs of SEQ ID NO 10 to SEQ ID NO 12 all have the PASQF modified sequence before the 3 rd cysteine from the N-terminal. In addition, the 17 th amino acid of the chemotactic peptide is mutated into leucine.

The modified chemokine peptides, analogs and/or fragments thereof of the invention can inhibit angiogenesis-related diseases. Angiogenesis-related diseases include, but are not limited to, inflammatory diseases, chronic rheumatoid arthritis and psoriasis, diseases associated with abnormal vascular invasion, and cell proliferative diseases such as diseases associated with tumors or cancers (e.g., prostate cancer, breast cancer, uterine cancer, blood cancer, ovarian cancer, endometrial cancer, cervical cancer, colorectal cancer, testicular cancer, lymphatic cancer, rhabdomyosarcoma, neuroblastoma, pancreatic cancer, lung cancer, brain cancer, skin cancer, stomach cancer, oral cancer, liver cancer, larynx cancer, biliary cancer, thyroid cancer, liver cancer, kidney cancer, and nasopharyngeal cancer, etc.).

The chemokine-modified peptide and/or the pharmaceutical composition containing the chemokine-modified peptide can be orally, parenterally, inhalatively, rectally, vaginally, intradermally, transdermally or topically administered, and a pharmaceutical unit can comprise a traditional nontoxic pharmaceutically acceptable carrier, adjuvant and vehicle.

The chemokine-modified peptide and/or the pharmaceutical composition comprising the same of the present invention can be administered once, multiple times within 24 hours or continuously. When the mode of injection is continuous administration, suitable conventional modes may be selected, including, but not limited to, intravenous drip, intravenous pump, implantable syringe pump, or topical administration. The duration of treatment can be adjusted as appropriate for different conditions, such as the course and severity of angiogenesis. The modified chemokine peptides of the invention alone or in combination with other agents of the invention are administered until cured, or for extended life.

In another embodiment, the invention provides a pharmaceutical composition for treating cancer and inhibiting tumor. The pharmaceutical composition comprises an effective amount of the chemokine-modified peptide or the analog thereof and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include solvents, dispersants, coatings, antibacterial/antifungal agents, isotonic, absorption delaying agents, and the like.

[ examples ] A method for producing a compound

1. Cell chemotaxis

Migration of LMVECs was assessed using Boyden chamber assay, and LMVECs were cultured in HuMedia-EB containing 2% FCS for 28 hours, followed by 12X10⁴ cells/cm²The cells of (2) were plated on polycarbonate filters (Sigma-Aldrich) with a pore size of 5 μm coated with 10. mu.g/ml of fibrinectin. 10ng/mL of CXCL8, CXCL6, CXCL1, CXCL5, and CXCL8-IP10 or IL8-F17LIP10, respectively, were placed at the bottom of the Boyden chamber. LMVECs were cultured in Boyden chambers at 37 ℃ for 4 hours, after which the filters were fixed and stained using Diff-quick (Harleco), and the number of migrated cells was counted using (HPFs) (X200).

Figure 3 shows that the number of migrations with the chemokine CXCL8 was the greatest.

In FIG. 4, the black part has only chemical hormone stimulation, the gray part has chemical hormone stimulation and CXCL8-IP10, and the white part has chemical hormone stimulation and IL8-17LIP 10. From FIG. 4, it is understood that CXCL8-IP10 and IL8-17LIP10 have the same effect of inhibiting cell migration, and IL8-17LIP10 has the better effect of inhibiting cell migration than CXCL8-IP 10.

2. Nude mouse xenograft analysis

Athymic males (BALB/c at 4-6 weeks) were placed in sterile procedure rooms. The room was aseptically operated with adequate drinking and feed, and mice were monitored daily. GFP-tagged PC-3 cells (PC-3-GFP) were injected into the right abdomen of 3 nude mice, each nude mouse injected 5X10⁶The cell of (1). After 2-4 weeks of culture, tumors were harvested for transplantation. Tumor tissues of the 3 nude mice were taken out and sectioned (1 mm)³Section (b) and transplanted into the prostate of other mice under anesthesia and sterilization. On day 5 after the transplantation, the animals were divided into 2 groups (12 animals per group) and injected subcutaneously with 100. mu.l of saline (control group) or the sequence of the present invention (IL8-17LIP10(SEQ ID NO:12), 0.5mg/kg) per day (experimental group) for 24 days. Images of tumor growth were taken at

day

12, 18, and 24 using an optical dissecting microscope and digital camera with a 515mm filter. Tumor volume was calculated as follows: tumor volume ═ width (length x)²)/2. On day 24, all mice were sacrificed and tumor GFP fluorescence images were taken. Microvessel density was calculated as follows: density-microvascular length/tumor volume. Tumor samples were fixed in 4% formaldehyde and embedded in paraffin for subsequent immunohistochemical analysis.

FIG. 5 shows the tumor volumes of the experimental and control groups at

days

12, 18 and 24. As shown in FIG. 5, the sequence of the present invention (IL8-17LIP10) significantly inhibited tumor growth, and inhibited tumor volume by more than 5 times, and the inhibitory effect was more significant with time.

FIG. 6 shows the tumor weights of the experimental and control groups at day 24. As is clear from FIG. 6, the sequence of the present invention (IL8-17LIP10) significantly inhibited tumor growth by more than 2 times the tumor weight.

3. Immunohistochemical analysis

Tumor sections embedded in paraffin were deparaffinized and rehydrated with PBS. In detail, the sections were wetted 3 times with PBS and heat-treated for 15 minutes in 10mM sodium citrate (pH 6.0). Thereafter, the cells were treated with 3% hydrogen peroxide for 10 minutes to remove endogenous peroxidase activity, washed with PBS 3 times, treated with a protein blocking solution (5% horse serum-containing PBS solution) at room temperature for 15 minutes, washed with PBS 3 times, and reacted with mouse anti-VEGF monoclonal antibody (1: 50), rabbit anti-NF-. kappa.B polyclonal antibody or goat anti-CD 3 polyclonal antibody (1: 50) at 4 ℃ for 20 hours. After completion of the reaction, the reaction mixture was washed 3 times with PBS and reacted with an appropriate secondary antibody at 37 ℃ for 40 minutes. After washing with PBS 3 times, the sections were reacted with biotin-labeled goat anti-mouse or anti-rabbit anti-goat IgG polyclonal antibody in a dark room for 30 minutes. In the chromogenic reaction, 3 washes with PBS, staining with diaminobenzidine solution for 10 minutes, and then staining with hematoxylin for 1 minute (Kollmar et al, 2007). The negative control group replaced the primary antibody with PBS. The Image was converted to gray scale (0-225), expressed as Integrated Optical Density (IOD), and the density was measured using Image-Pro 6.0Microsoft software. Transplanted tumors were analyzed in 5 mice per group. Randomly select 5 slice images per tumor to average out the gray scale value of each tumor (Csillik et al, 2005). Immunohistochemical analysis of CD31 can be used to identify microvessel densities, counted using Image-Pro 6.0Microsoft software, and expressed as mean values.

Fig. 7 shows the microvascular density of the control and experimental groups. As shown in FIG. 7, the sequence of the present invention (IL8-17LIP10) was found to be effective in inhibiting the microangiogenesis of prostate cancer in mice.

C57BL/6 mouse assay

C57BL/6 mice were placed in sterile operating rooms. The room was aseptically operated with adequate drinking and feed, and mice were monitored daily. LLW2 cells (Lewis lung cancer) were injected into the right abdomen of 3 nude mice, each nude mouse injected 5X10⁶The cell of (1). After 2-4 weeks of culture, tumors were harvested for transplantation. A total of 24 mice received tumor transplants. On day 5 after transplantation, animals were divided into 4 groups (6 animals per group), group A was injected 4 times per worship with IL8-IP 10500 ug/kg, group B was injected 2 times per worship with IL8-IP 10500 ug/kg, group C was injected 2 times per worship with IL8-IP 10250 ug/kg, and group D was subcutaneously injected every dayNext, 100. mu.l of saline was injected. Tumor volume was calculated as follows: tumor volume ═ width (length x)²)/2. The length and width were measured using Image-Pro 6.0Microsoft Windows. On day 24, all mice were sacrificed (sacrificed) and tumor GFP fluorescence images were taken. Microvessel density was calculated as follows: density-microvascular length/tumor volume. Tumor samples were fixed in 4% formaldehyde and embedded in paraffin for subsequent immunohistochemical analysis.

Figure 8 shows that group a had the best effect in inhibiting tumor volume. On day 24, before sacrificing all mice, the tumor sizes were measured and it was found that the tumor volume of mice in group A injected 4 times per worship CXCL8-IP 10500 ug/kg was 30% smaller than that of the tumors in group D.

FIG. 9 shows sacrificed lung tumor tissue in mice, and it can be seen from A that distant metastasis of tumor has been significantly inhibited by CXCL8-IP 10. A in FIG. 9 is lungs taken out of mice injected with CXCL8-IP 10500 ug/kg 4 times per worship after sacrifice on day 24; panel B shows the lungs of mice administered saline, wherein white tumor sites indicated by arrows are foci of metastasis to the lungs. The lungs of mice administered CXCL8-IP10 were very clean and no metastatic lesions appeared in the lungs.

5. Neutrophil chemotaxis assay

Neutrophil chemotaxis was assessed by a modified Boyden chamber microchemical assays. Leukocytes are obtained from human peripheral blood by normal concentration gradient separation, whereas neutrophils are obtained from the bottom of the low gradient separation and cleared of contaminating erythrocytes using hypotonic lysis. Inducing stirring of purified 5x10⁶The neutrophils are suspended in HBSS (400mg/L potassium chloride (KCl), 60mg/L potassium hydrogen phosphate (KH)₂PO₄) 8000mg/L sodium chloride (NaCl), 350mg/L sodium bicarbonate (NaHCO)₃) 90mg/L sodium hydrogen phosphate (NaH)₂PO₄·7H₂O), 1000mg/L glucose (glucose), and 0.5% bovine placental serum (total calf serum; pH 7.4), followed by treatment of neutrophils with Calcein AM (Invitrogen, Stockholm, S)weden) for 30 minutes at 37 ℃. Chemokines (e.g., CXCL 820 ng/mL) alone or in combination with other anticoagulants (IL8-IP10F17L, etc.) were placed at the bottom of the culture zone of the Boyden chamber and purified neutrophils were placed at the upper end of the culture chamber, separated from the bottom by a 5 μm pore size polycarbonate filter. After incubation at 37 ℃ and 5% carbon dioxide (CO2) for 30 minutes, the non-mobilized cells and the filters were removed and the mobilized cells were disrupted and analyzed by VICTOR3(Perkin-Elmer, UK, circulation: 485 nm; emission:530nm), the percentage of cells mobilized being expressed as chemotactic Index (Chemotaxis Index (CI) value):

CI＝(intensity_antagonist-intensity_HBSS)/(intensity_CXCL8-intensity_HBSS)X100％

wherein intensity_antagonistRefers to the cell movement generated by adding the protein with antagonistic effect and CXCL8 at the same time; intensity_CXCL8Means that the cells are moved only by CXCL8 stimulation, and the intensity_HBSSIt is meant that the movement of the cells is caused by gravity.

RT-PCR analysis of CXCR1/2 and CXCL8 gene expression levels in individual tumor cells.

RNA of each cancer cell line in this experiment was extracted by TRIzol reagent (Invitrogen, America) and its instructions, and most of the RNA was quantified using

ND-1000 spectrophotometer. During the reverse transcription reaction, the sample was prepared using cDNA reverse-transcription kit (Prime Script)^TMRT reagent kit, Takara, Japan) and placed on ice prior to gene expression analysis. GAPDH is an internal control group. RT-PCR was performed by SYBR (Premix Ex Taq)^TMTakara, Japan), the CXCL primers of which are as follows:

5′-gagcactccataaggcacaaa-3′(forward)and 5′-atg gttccttccggtggt-3′(reverse)for CXCL8；

5′-gaccaacatcgcagacacat-3′(forward)and 5′-tgcttgtctcgttccacttg-3′(reverse)for CXCR1；

5′-ggctaagcaaaatgtgatatgtacc-3′(forward)and 5′-caaggttcgtccgtgttgta-3′(reverse)for CXCR2。

method for calculating gene expression expressed by expression gene expression 2^-ΔΔCt

Epimedium I, tumor cell strain

HCC827	lung adenocarcinoma
		HCC827GR	HCC827 gefitinib-resistant
H1975	lung adenocarcinoma
		H2170	lung squamous cell carcinoma
H157	oral squamous cell carcinoma
		CT26	colon carcinoma cell
CL1-0	lung adenocarcinoma
		CL1-5	lung adenocarcinoma
PC9	lung adenocarcinoma
		H3255	Non small cell lung carcinoma
A549	lung adenocarcinoma
		H520	lung squamous cell carcinoma
H460	Non small cell lung carcinoma

FIG. 10 shows that RT-PCR is used to detect that some tumor cells have high expression of the chemokine receptor CXCR1, and many tumor cells express high amount of CXCL8 which is the receptor of two types such as CXCR1/2, so that CXCR1/2 receptor antagonist can be used to prevent tumor cell growth and metastasis.

FIG. 11 shows that more anti-cancer agents are designed, and the addition of the mutation of amino acids No. 12 or/and No. 13 or/and No. 17 to CXCL8-IP10 disclosed previously can enhance the anti-cancer effect on the physiological response triggered by the receptor CXCR1/2, effectively achieve the inhibition of tumor proliferation, drug resistance, metastasis and angiogenesis caused by the tumor cells expressing CXCR1/2 receptor or the tumor cells expressing the receptor-responsive cytokines (CXCL1,2,3,5,6,7,8, etc.) with high expression.

Therefore, the chemokine peptide can effectively inhibit the growth of tumor and angiogenesis and treat cancer.

All the technical features of the invention disclosed in the specification may be combined in any manner. Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, all features disclosed herein are only examples of a generic series of equivalent or similar features.

From the foregoing, it will be appreciated by those skilled in the art that the foregoing and other features of the invention, which are set forth in the following description, are susceptible to various modifications and alternative uses without departing from the spirit and scope of the invention.

Sequence listing

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Leu His Pro Lys Phe Ile Lys Glu Leu Arg Val Ile Pro Ala Ser Gln

20 25 30

Phe Cys Ala Asn Thr Glu Ile Ile Val Lys Leu Ser Asp Gly Arg Glu

35 40 45

Leu Cys Leu Asp Pro Lys Glu Asn Trp Val Gln Arg Val Val Glu Lys

50 55 60

Phe Leu Lys Arg Ala Glu Asn Ser

65 70

<210> 11

<211> 71

<212> PRT

<213> Artificial sequence

<220>

<223> modified ELR-CXC chemokines

<400> 11

Ser Ala Lys Glu Leu Arg Cys Cys Ile Arg Thr Tyr Ser Lys Pro Leu

1 5 10 15

His Pro Lys Phe Ile Lys Glu Leu Arg Val Ile Pro Ala Ser Gln Phe

20 25 30

Cys Ala Asn Thr Glu Ile Ile Val Lys Leu Ser Asp Gly Arg Glu Leu

35 40 45

Cys Leu Asp Pro Lys Glu Asn Trp Val Gln Arg Val Val Glu Lys Phe

50 55 60

Leu Lys Arg Ala Glu Asn Ser

65 70

<210> 12

<211> 72

<212> PRT

<213> Artificial sequence

<220>

<223> modified ELR-CXC chemokines

<400> 12

Gly Ser Lys Glu Leu Arg Cys Gln Cys Ile Arg Thr Tyr Ser Lys Pro

1 5 10 15

Leu His Pro Lys Phe Ile Lys Glu Leu Arg Val Ile Pro Ala Ser Gln

20 25 30

Phe Cys Ala Asn Thr Glu Ile Ile Val Lys Leu Ser Asp Gly Arg Glu

35 40 45

Leu Cys Leu Asp Pro Lys Glu Asn Trp Val Gln Arg Val Val Glu Lys

50 55 60

Phe Leu Lys Arg Ala Glu Asn Ser

65 70

Claims

1. A pharmaceutical composition comprising a chemokine-modified peptide for treating cancer and inhibiting tumors, comprising a chemokine-modified peptide and a pharmaceutically acceptable excipient;

the chemokine modified peptide is IL8-IP10T12S, and the unmodified precursor of the modified chemokine peptide is from the source chemokine peptide SEQ ID NO. 1;

the chemokine-modified peptide is characterized by having an N-terminal (N'), and comprises: (a) signature a, wherein said signature a is glutamic acid (E) -leucine (L) -arginine (R), is located at the N-terminus of said chemokine-modifying peptide; (b) a characteristic sequence B, wherein the characteristic sequence B is proline (P) -alanine (A) -serine (S) -glutamine (Q) -phenylalanine (F), and is the 3 rd cysteine (Cys) from the N end of the chemokine modified peptide³) (ii) a Wherein the chemokine modified peptide comprises the characteristic sequences of (a) and (b) and has a modification position, and the modification position is located at 12 th amino acid counted from N end on the source chemokine peptide SEQ ID NO:1 and is replaced by serine (S) through threonine (T);

the chemokine-modified peptides are useful for treating cancer or inhibiting tumor growth.

2. The pharmaceutical composition of claim 1, wherein the cancer or tumor comprises prostate cancer, pancreatic cancer, lung cancer.

3. The pharmaceutical composition of claim 2, wherein said cancer or tumor is characterized by tumor cells having expression of CXCR1/2 or having high levels of CXCL8 chemokine or receptor thereof.