CN108640970B

CN108640970B - Polypeptide targeting ectopic ATP5B pathway and application thereof

Info

Publication number: CN108640970B
Application number: CN201810270410.6A
Authority: CN
Inventors: 王宜强; 王婷; 林丹丹
Original assignee: First Affiliated Hospital of Suzhou University
Current assignee: First Affiliated Hospital of Suzhou University
Priority date: 2018-03-29
Filing date: 2018-03-29
Publication date: 2021-07-23
Anticipated expiration: 2038-03-29
Also published as: CN108640970A

Abstract

The invention provides a polypeptide targeting an ectopic ATP5B pathway and application thereof, wherein the motif is NPLKXDWG, wherein X is valine V or methionine M, and the N end and/or C end of the motif are/is connected with 0 or 2 amino acids; the two amino acids which are sequentially connected at the N end are proline P and valine V, and the two amino acids which are sequentially connected at the C end are leucine L and proline P. The invention also provides the application of the polypeptide in preparing tumor diagnosis reagent and tumor targeting medicine. The polypeptide provided by the invention can influence the aggregation function of platelets and reduce the formation of a membrane glycoprotein GPIIb/IIIa complex. The peptide segment has binding effect on part of tumor cells, and the polypeptide connecting toxic sequence complex can inhibit the proliferation of tumor cells expressing ectopic ATP5B under certain concentration. Therefore, the polypeptide provides important theoretical and practical basis for early diagnosis and targeted therapy of tumors, and has wide application prospect.

Description

Polypeptide targeting ectopic ATP5B pathway and application thereof

Technical Field

The invention relates to a targeting polypeptide, in particular to a polypeptide targeting an ectopic ATP5B pathway and application thereof.

Background

Malignant tumors (including leukemia) are currently an important disease that endangers human health, and a significant portion of patients fail various therapies, although many advances have been made in biotherapy (including cell therapy, immunotherapy, etc.) and even targeted therapy in recent years after undergoing surgical treatment, radiotherapy, chemotherapy. In cases with significant effects in various initial treatments, it is also an important issue to be solved in tumor treatment to take appropriate measures to prevent metastasis or recurrence. The development of more efficient or less expensive antitumor drugs has been a target sought by scientists and medical workers in this field.

At least 50 years ago, scholars were aware that platelets play an important role in metastasis of malignant tumors: after entering blood vessels, solid tumor cells form aggregates (PTA) with platelets in the blood; PTA formation favors tumor metastasis, while anti-platelet measures can inhibit tumor metastasis. When studying the mechanism of PTA formation, we firstly discover and prove that ATP5B (ATP synthase beta subbunit, ATP synthase beta subunit, is distributed only inside the inner mitochondrial membrane under 'normal' condition) expressed at the surface of partial tumor cell membrane in an ectopic way interacts with glycoprotein IIb (GPIIb) on the surface of platelet and participates in mediating PTA formation; furthermore, a sequence peptide fragment from GPIIb can be efficiently combined on ATP5B on the surface of a tumor cell; the polypeptide is used as a targeting peptide, and a known toxic polypeptide can be guided to tumor cells expressing ectopic ATP5B, so that the targeted killing of the tumor cells is realized. Meanwhile, angiogenesis is often accompanied in solid tumors, and the existing data show that endothelial cells of the new blood vessels also express ectopic ATP5B, while the surfaces of the endothelial cells of the normal steady blood vessels do not express ectopic ATP5B, so that the targeted polypeptide can also realize targeted killing on the new blood vessels.

The research progress at home and abroad is briefly described and analyzed as follows:

1. for tumor targeted therapy

The concept and attempt of "targeted therapy" was first in the 1980 s with monoclonal antibody technology and has become increasingly appreciated with the popularity of monoclonal antibody drugs. However, the accumulated data show that a Specific molecule, i.e. Tumor-Specific Antigen (Tumor-Associated Antigen), expressed only in Tumor cells is not present, while most Tumor-Associated antigens (Tumor-Associated antigens) are also expressed in normal cells, so that the killing range of antibodies "targeting" these molecules is far beyond that of Tumor cells, for example, currently commonly used Rituximab can eliminate lymphoma cells highly expressing CD20 and B lymphocytes at normal development stage also expressing CD20, thereby causing immunosuppression of the whole line. Similar limitations exist with the recent emergence of chimeric antigen receptor T cell (CAR-T) therapies, such as various CARs-19 against CD 19. Therefore, at the present stage, there is still a need to find new tumor-targeted therapeutic schemes or strategies by combining the characteristics of tumor cells and mature biotechnology.

Targeted therapy is an essential feature of modern drug therapy, and is a key area of drug development, particularly in the treatment of all tumors, including hematological tumors. The classification of the action targets of chemical drugs and biological drugs applied to the treatment of hematological malignancies (which are crossed with each other) is combined, and the application or the current research situation and the development dynamics of targeted therapy are analyzed:

2. main target point for tumor target therapy

The targets of tumor targeted therapy, whether applied clinically, under development, or still in the fundamental research stage, are currently largely divided into three categories: (1) tumor cell surface antigen: the medicines or candidate medicines aiming at the target points are mainly monoclonal antibody preparations or derivatives thereof (including Fab fragments, humanized modifiers and coupled substances with other components) and directly attack and kill tumor cells carrying specific antigens. Typical examples include: the anti-CD 20 natural monoclonal antibody or humanized monoclonal antibody Ofatumumab, antibody drug Brentuximab vedotin formed by CD30 monoclonal antibody and vedotin, and bispecific antibody aiming at double targets. The latter can simultaneously recognize tumor cell surface antigen and CTL surface antigen, connect tumor cells and killer cells together and activate the latter, thereby improving killing efficiency or overcoming tumor cell immune escape mechanism. anti-CD 19/CD3 diabody, anti-CD 33/CD 3 diabody, anti-CLL 1/CD3 diabody, anti-CD 30/CDl6a diabody, etc. are also in different research stages, respectively. (2) Tumor closely related signaling pathway molecules: the medicines in the class mainly pass through the pairCellsThe various signal pathways of the Btk inhibitor Ibrutinib, the PKCl3 inhibitor Enzastaurin, the PI3K inhibitor Iderlalisib or the small molecule inhibitor AMG319, the Bcl.2 inhibitor ABT0199/ABT-199, the mTORCl inhibitor Everolimus, the PI3K isomer and mTORCl and the TORC2 inhibitor SAR 2450 4096 and the like, and the MDM2 antagonist RG7112 and the like. (3) Genetic material of tumor cells: genetic material alterations (including sequence mutations or epigenetic modifications) are one of the material bases for tumorigenesis. Although it is theoretically possible to restore normal tumor cells by correcting mutated genetic material, it is not clinically feasible due to technical limitations; the epigenetic inheritance can be adjusted by medicines, for example, pandeacetylase inhibitor Panobinostat and other chemotherapeutic drugs (ifosfamide, carboplatin and etoposide) are used for treating relapsed refractory Hodgkin lymphoma, so that a primary effect is achieved.

In addition, there are targeted therapies directed against the body's anti-tumor immune cells, or against the tumor cell microenvironment, such as antibody or other means to inhibit the T cell PD-L1/PD-1 interaction to restore the anti-tumor activity of T cells, and so on, which are not directly applied to tumor cells, and are omitted here.

3. Possible application of platelet-tumor aggregation mechanism target in tumor prevention and treatment

3.1 role of platelet-tumor aggregation in tumor metastasis: hematogenous metastasis is the major pathway of tumor distant metastasis. The existence of platelet-tumor aggregation was recognized as early as a century ago^[1]By the 1960 s, the importance of this mechanism in tumor metastasis was recognized and started to prevent tumor metastasis by interfering with this mechanism^{[2, 3]}. Platelets bound to the surface of tumor cells are traditionally considered to have a promoting effect on metastasis, and an anti-platelet strategy is clinically applied to prevent tumor metastasis. The mechanism by which platelets promote tumor metastasis includes: direct 'umbrella' action on tumor cells; increase the viscosity of tumor cells or cell clusters and facilitate the gathering to the vessel wall; mediate the combination with endothelial cells, release proteases and degrade the matrix or basement membrane between endothelial cells so as to facilitate the emigration of tumor cell masses. The 'umbrella protection' mechanism is characterized by at least four factors: the tumor cells are protected from being monitored and attacked by immune cells through a physical barrier mechanism, so that the survival and the metastasis of the tumor cells in the blood phase are facilitated^{[4, 5]}(ii) a Interfere with the killing effect of certain cytokines on tumor cells; platelets transfer MHC-class I molecules from their surface to tumor cellsSurface, rendering it "disguised" as normal cells, thereby evading recognition and killing by immune cells^[6](ii) a The platelet induces the epithelial-mesenchymal transition of the combined tumor cells through direct action, thereby promoting the metastasis and leading to the construction of a microenvironment niche suitable for the implantation and expansion of a metastasis focus "^{[7, 8]}. Although there is much evidence that platelets play a "culprit" role in tumor metastasis, there is also some evidence that platelets bound to tumor cells may be detrimental to the survival or proliferation of tumor cells. For example, platelets can induce apoptosis or directly kill tumor cells, whether against solid tumor cells entering the blood or leukemia cells originating from the blood^{[9, 10]}(ii) a However, the sensitivity of various cells to the killing effect of platelets is inconsistent, and Sagawa et al report that platelets can directly kill K562, KU812, LU99A, KGl tumor cells, but are ineffective against U937, MIA, PaCa-2, MOLT-4 tumor cells^[11]. Our own earlier work also found that platelets in physiological range numbers inhibited proliferation of various tumor cells to varying degrees^[12]More recently, it has been demonstrated that platelets that bind to leukemia cells can alter the sensitivity of leukemia cells to chemotherapeutic drugs^[13]. The "net" effect of platelets on the tumor cells with which they come into contact is therefore dependent on a balance of two effects, such as the recent demonstration by German scholars in a mouse tumor model that platelets bind to B16 tumor cells and promote tumor entrapment in the lung, but at the same time also inhibit tumor cell proliferation^[14]。

3.2 platelet-tumor aggregation mechanism: about 10 pairs of molecules are known to participate in PTA formation, the most important of which are various glycoproteins on the membrane, and sometimes the same molecule (such as GPIIbIIIa) on two cell membranes, linked by another ligand (such as fibrin). Although the molecular species on the surface of platelets are relatively fixed, the molecular pattern of the tumor cell membrane surface involved in PTA formation may vary depending on the tissue origin, differentiation status, and even individual differences, wherein there may be either a universally expressed molecule such as gpiibia or a molecule expressed only in a portion of the tumor. If a molecule is involved in PTA formation and belongs to tumor-associated or even tumor-specific antigens (i.e., is not expressed or is low expressed in normal tissues), it is likely to become a target molecule for preventing and treating tumor metastasis by interfering with PTA mechanism. This is the case for the topic group selection ectopic ATP 5B.

3.3 ectopic expression of ATP5B on tumor cell membranes: the "universal currency" for cellular energy metabolism is ATP, and the main enzyme catalyzing the synthesis of ATP is F1F0-ATP synthase, located in the inner mitochondrial membrane. The enzyme consists of 24 proteins in total, and ATP5B is three beta subunits of F1. Typically, the enzyme is distributed only in the inner mitochondrial membrane. However, Das first reported in 1994 that ATP5B protein is also present on the cell surfaces of K562 and A439 cells and is recognized by lymphocyte NK or LAK cells^[15]. The presence of ATP5B was subsequently detected on the cell membranes of other tumors, for example Dowling compared membrane proteins from two clones of MDA-MB-435S female parent but with different invasiveness, and 16 proteins were found to be up-regulated in expression of highly invasive clones, including ATP5B^[16](ii) a The Liyilei topic group at Jilin university finds that the expression level of ectopic ATP5B in the high-metastatic prostate cancer cell strain PC-3M is higher than that of the parental strain PC-3 cell^[17](ii) a The Weiyuquan and Yangjingliang subjects demonstrated that the expression ratios of ectopic ATP5B in non-small cell lung cancer and paracarcinoma tissues were 72% (23/32 cases) and 26% (16/62), respectively, while the ratios in squamous carcinoma and small cell lung cancer were 67% (22/33 cases) and 0% (0/10), respectively^[18](ii) a The high challenge group demonstrated that ATP5B on the surface of tumor cell membrane is transported to the tumor cell membrane from the inner mitochondrial membrane, but may not be related to the malignancy of the tumor^[19](the universality of this conclusion is questionable, since the number of tumor cells used in this article for comparison of malignancy is too low and the sources are different, e.g. where the two "highly malignant" cell lines are lung cancer 95-D and liver cancer HepG2, respectively, and the comparability of lung cancer A549 and liver cell line L-02, both "low malignancy" is poor^[19]) (ii) a The Yangbulin topic group finds that the loss or low expression of ectopic ATP5B expression is a risk factor of large tumor volume, high TNM classification, lymphatic metastasis and paratopic invasion in 126 clinical gallbladder cancer cases^[20]。

In addition to tumor cells, it is now known that other cells may also express ectopic ATP 5B-If tumor surface ectopic ATP5B is to be used as a therapeutic target, other cells expressing ectopic ATP5B will be targets for side effects, and thus particular attention is required. Consistent with tumor therapy target screening, although it is currently known that vascular endothelial cells express ectopic ATP5B, the current evidence is that vascular endothelial cells cultured in primary culture or in vitro and in logarithmic growth phase are thin rather than in situ vascular endothelial cells in vivo; it is now demonstrated that ectopic ATP5B on vascular endothelial cells can act as angiostatin^[21]、kringle 1–5 （K1–5）^[22]And fragment epithi μ M-derived factor, namely PEDF^[23]And binding of these ligands to ATP5B results in inhibition of endothelial cell proliferation and even apoptosis. If ectopic ATP5B is expressed only on proliferating vascular endothelial cells and on non-proliferating vascular endothelial cells, the neovascular ATP5B in tumors will serve as an additional target for tumor therapy without causing side effects on normal blood vessels, as in the anti-neovascular strategies of previous tumor therapy. It has been shown that PEDF, which was previously known to target neovascular endothelial cells, also binds to ectopic ATP5B on the surface of tumor cells and inhibits apoptosis of tumor cells^[24]It is suggested that ATP5B ectopically expressed in different cells may mediate the same pathway and cause similar effects when bound by a ligand.

Disclosure of Invention

The invention aims to provide a polypeptide targeting an ectopic ATP5B pathway and application of the polypeptide in preparing a tumor diagnostic reagent and a tumor targeting medicament.

The motif of the polypeptide targeting the ectopic ATP5B pathway is NPLKXDWG, wherein X is valine V or methionine M, and the N end and/or C end of the motif are connected with 0 or 2 amino acids.

Furthermore, the two amino acids sequentially connected at the N end are proline P and valine V, and the two amino acids sequentially connected at the C end are leucine L and proline P.

The amino acid sequence of the polypeptide is shown as SEQ ID NO.1-SEQ ID NO.8, and is specifically shown as follows:

N2G：NPLKVDWG，

N2Gm：NPLKMDWG，

N2G-LP：NPLKVDWG-LP，

PV-N2G：PV-NPLKVDWG，

PV-N2G-LP：PVNPLKVDWGLP，

N2Gm-LP：NPLKMDWG-LP，

PV-N2Gm：PV-NPLKMDWG，

PV-N2Gm-LP：PVNPLKMDWGLP。

the invention relates to application of a polypeptide targeting an ectopic ATP5B pathway in preparing a tumor diagnostic reagent.

The invention relates to application of a polypeptide targeting an ectopic ATP5B pathway in preparing a tumor targeting medicament.

A tumor targeting therapeutic agent comprising a polypeptide of the invention targeting the ectopic ATP5B pathway.

Preferably, the polypeptide targeting the ectopic ATP5B pathway is conjugated to an active molecule comprising a toxic molecule having a tumor therapeutic effect.

Preferably, the polypeptide targeting the ectopic ATP5B pathway is coupled to a drug delivery carrier, and the drug delivery carrier is a nanoparticle or a microsphere.

Preferably, the polypeptide targeting the ectopic ATP5B pathway is expressed on a recombinant toxin molecule, and the recombinant toxin molecule is formed by expressing a recombinant gene after gene recombination between a DNA sequence corresponding to the polypeptide targeting the ectopic ATP5B pathway and a DNA sequence of a protein toxoid molecule.

Has the advantages that: the invention provides a polypeptide of a targeted ectopic ATP5B access and application thereof, the invention utilizes a monoclonal antibody (SZ-22) of mouse anti-human platelet membrane glycoprotein GPIIb to carry out three rounds of biological affinity elutriation screening on a random 12 peptide library displayed by phage, extracts DNA and determines a sequence after amplifying a large number of obtained clones, and then compares the determined phage monoclonal sequence to obtain a peptide segment sequence N2G: NPLKDVDWG, after artificial synthesis, is proved by a flow cytometry technology and a Western blot technology that the peptide segment N2G can block the combination of a monoclonal antibody (SZ-22) of mouse anti-human platelet membrane glycoprotein GPIIb and normal human platelets. Meanwhile, the peptide segment can influence the aggregation function of platelets and reduce the formation of a membrane glycoprotein GPIIb/IIIa complex. The peptide segment has a binding effect on partial tumor cells, and a composite polypeptide (NPLKDVDWG-LP-KLAKLAKKLAKLAK) which is formed by taking N2G as a targeting peptide and a known toxic peptide segment KLAKLAKKLAKLAK can inhibit the proliferation of the tumor cells under a certain concentration, so that the N2G polypeptide has a wide application prospect in the aspect of preparing tumor diagnosis reagents and tumor targeting drugs.

Drawings

FIG. 1 shows the homology alignment of 11 PhD-selected polypeptides and the alignment of the sequences with the CD41 gene (in the PhD12 vector used, the three amino acids immediately after the 12 peptide are GGG, so the position g is complemented after the three polypeptides H10, A11 and B10).

FIG. 2 is a graph showing the blocking effect of a flow-type detection polypeptide on the binding of a mouse anti-human platelet membrane glycoprotein GPIIb monoclonal antibody SZ-22 to platelets, wherein A, B shows that the concentration of the corresponding SZ-22 antibody is 0.1 μ g; C. the concentration of SZ-22 antibody in the D-plot was 1. mu.g.

FIG. 3 is a graph showing the effect of Western blot on the detection of the binding of the polypeptide to the total platelet protein and SZ-22 monoclonal antibody of mouse anti-human platelet membrane glycoprotein GPIIb, in which A, B indicates that the concentration of the SZ-22 antibody is 0.1. mu.g; C. the concentration of SZ-22 antibody in the D-plot was 1. mu.g.

Fig. 4 is a graph showing the results of inhibition of ADP-induced platelet aggregation by the N2G polypeptide (Peptide 8) (aggregation rate was calculated from the platelet aggregation graph).

FIG. 5 inhibition of ADP-induced activation by N2G polypeptide (Peptide 8) results are shown (platelet activation is shown by PAC-1 staining).

FIG. 6 is a graph showing the binding strength of FITC-N2G polypeptide and Peptide S to four cells detected by flow cytometry.

FIG. 7 is a fluorescence micrograph of four cells bound to a FITC-N2G polypeptide.

FIG. 8 is a graph comparing the maximum inhibitory concentrations of Peptide 1 and Peptide 5.

FIG. 9 is a graph showing the effect of Peptide 1 and Peptide 5 on the proliferation of seven tumor cells.

Detailed Description

The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1: obtaining a polypeptide sequence targeting the ectopic ATP5B pathway

The purified monoclonal antibody (SZ-22) of mouse anti-human platelet membrane glycoprotein GPIIb and 10 are mixed¹⁰ pfu phage display 12 peptide library (New England Biolabs, embedding random peptide with 12 amino acids in pIII protein of M13 phage) incubation for 10-60 min at room temperature, TBST washing off unbound phage, eluting phage bound with SZ-22 antibody with glycine eluent, amplification titration, 2 rounds of panning and screening, selecting 12 phage single clones for DNA extraction and sequence determination, translating the obtained nucleotide sequence into polypeptide, carrying out homology analysis on the polypeptide sequence corresponding to phage clone by NCBI database and Clustal software, finding that 11 peptide segments are highly homologous with each other and with human CD41 protein (GPIIb), and the alignment sequence is: NPLKVDWG, designated N2G polypeptide. (see FIG. 1). Respectively extending two amino acids forwards and/or backwards according to the sequence of GPIIb corresponding to the GPIIb, or changing V in the two amino acids into M, respectively forming the following polypeptide sequences:

N2G：NPLKVDWG；

N2Gm：NPLKMDWG；

N2G-LP：NPLKVDWG-LP；

PV-N2G：PV-NPLKVDWG；

PV-N2G-LP：PVNPLKVDWGLP；

N2Gm-LP：NPLKMDWG-LP；

PV-N2Gm：PV-NPLKMDWG；

PV-N2Gm-LP：PVNPLKMDWGLP。

example 2: binding of synthetic polypeptide fragments to platelets

The polypeptide was chemically synthesized according to the sequence of N2G. Platelets were prepared from normal human peripheral blood and adjusted to appropriate concentrations, and different concentrations (100. mu.M, 10. mu.M, 1. mu.M, 0.1. mu.M, 0.01. mu.M and 0. mu.M) of N2G polypeptide (Peptide 8) were incubated with 0.1. mu.g/1. mu.g of SZ-22 antibody, respectively, at room temperature for 30-60 min; adding platelets, incubating at room temperature for 30min, adding MTB (Modified Tyrode' S buffer) buffer, washing, adding the MTB buffer to resuspend the platelets, adding a PE-labeled goat anti-mouse IgG antibody as a secondary antibody, incubating at room temperature for 30min in the absence of light, adding the MTB buffer to wash, resuspending the platelets with the MTB buffer, and using a computer to detect the blocking degree of the polypeptide on the SZ-22 antibody and the platelets, wherein the random polypeptide Peptide S group and the blank group without the polypeptide are used as negative controls (see figure 2), and the result shows that the blocking effect of the polypeptide on the SZ-22 antibody and the platelets is more obvious along with the increase of the concentration of the N2G polypeptide compared with the blank group without the N2G polypeptide and the random polypeptide Peptide S group, and the blocking effect of the polypeptide on the SZ-22 antibody bound on the surfaces of the platelets in a low concentration but saturated manner is more obvious.

Extracting total normal human platelet protein, performing protein SDS-PAGE electrophoresis, respectively incubating polypeptide Peptide 8 and 0.1 μ g/1 μ gSZ-22 antibodies at different concentrations (100 μ M, 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M and 0 μ M) at 4 ℃ for 30-60min, incubating the mixture of the polypeptide and SZ-22 antibody as a primary antibody with platelet protein strips, incubating the primary antibody with goat anti-mouse IgG antibody marked by HRP as a secondary antibody, taking beta-Tubulin protein as an internal reference, observing the blocking degree of the combination of SZ-22 antibody and platelets by exposure and development, and taking a random polypeptide Peptide S group and a blank group without polypeptide as negative controls. The results (see FIG. 3) show that the blocking effect of the polypeptide on the binding of SZ-22 antibody to platelets is more significant and shows a dose-dependent effect with increasing polypeptide concentration compared with the blank group without the polypeptide and the random polypeptide Peptide S group, and the blocking effect of the polypeptide is significantly higher in the presence of low concentration of SZ-22 antibody than in the presence of high concentration of SZ-22 antibody.

Example 3: effect of N2G Polypeptides on platelet activation

1) Effect of Polypeptides on platelet aggregation function

Detecting by a platelet aggregation instrument: extracting normal human blood platelets and adjusting to a proper concentration, incubating the blood platelets and 10 mu M Peptide 8 polypeptide at room temperature for 30min, using an MTB buffer solution for a platelet aggregation instrument to adjust to zero, using blank group blood platelets without the polypeptide and blood platelets incubated by adding random Peptide S group as negative control, using 10 mu M ADP in a 37 ℃ aggregation instrument to stimulate the blood platelets to aggregate, and observing the degree of the blood platelet aggregation by using the platelet aggregation instrument. The results (see fig. 4) show that 10 μ M polypeptide was able to reduce ADP-stimulated platelet aggregation compared to the blank and the randomized polypeptide Peptide S group without added polypeptide.

2) Effect of Polypeptides on the formation of the platelet membrane glycoprotein GPIIb/IIIa Complex

Flow cytometry detection: extracting normal human blood platelet and adjusting to proper concentration, incubating the blood platelet with 10 μ M Peptide 8 polypeptide at room temperature for 30min, incubating at 37 deg.C for 30min without adding ADP stimulating group and with 10 μ M ADP stimulating group, adding PAC-1 antibody for staining for 30min, and detecting the formation of membrane glycoprotein GPIIb/IIIa complex on computer. The results (see FIG. 5) show that 10. mu.M polypeptide can reduce the formation of the membrane glycoprotein GPIIb/IIIa complex caused by platelet stimulation with ADP, compared to the blank group without the addition of polypeptide, the group without the addition of ADP stimulation and the random polypeptide Peptide S group.

Example 4: interaction of N2G polypeptide with tumor cells

The N2G polypeptide has high homology with platelet membrane glycoprotein GPIIb (CD 41 protein), so that the polypeptide also mediates the interaction between platelets and tumor cells, has binding effect on part of the tumor cells, and can inhibit the proliferation of the tumor cells under a certain concentration by the composite polypeptide (NPLKDVDWG-LP-KLAKLAKKLAKLAK).

1) Binding capacity of N2G polypeptide to tumor cells

Tumor cells (K562, NB 4) as well as normal skin cells (HACAT) and skin cancer cells (A431) were incubated with 10. mu.M of artificially synthesized Peptide 8 polypeptide with FITC label at 4 ℃ for 30min, PBS was used to wash away unbound polypeptide molecules, and the average fluorescence intensity of N2G polypeptide bound to various cells was measured by flow cytometry. Tumor cells (K562, NB 4) and normal skin cells (HACAT) and skin cancer cells (A431) were incubated with 10. mu.M of synthetic Peptide 8 with FITC label at room temperature for 30min, PBS was used to wash away unbound polypeptide molecules, and immunofluorescence was used to detect the binding strength of N2G polypeptide to various cells. The results (see fig. 6, 7) show that the mean fluorescence intensity of N2G polypeptide binding to the tumor is increased compared to the control cells (HACAT, a 431) (fig. 6 shows that the random polypeptide Peptide S group is not different from the target polypeptide Peptide 8 group, since the amino acid composition of the random polypeptide and the target polypeptide is the same, but the position of the amino acid forming the Peptide segment is changed, which may indicate that the tumor cells have not very high requirements for the amino acid sequence of the polypeptide).

2) Median inhibitory concentration of the Complex polypeptide

Artificially synthesizing a composite polypeptide Peptide 1 (NPLKDVDWG-LP-KLAKLAKKLAKLAK) and a control group polypeptide Peptide 5 (KLAKLAKKLAKLAK) on the basis of the screened polypeptides displayed by phage, measuring the half inhibitory concentration (IC50), namely detecting the inhibitory action of the polypeptides (200 mu M, 160 mu M, 120 mu M, 80 mu M, 40 mu M, 10 mu M, 5 mu M, 1 mu M, 0.1 mu M, 0.01 mu M and 0 mu M) with different concentrations on acute promyelocytic leukemia cell NB4 by adopting the MTT method, and adding a polypeptide group as a control group. The results (see FIG. 8) show that Petide 1 has an IC50 of 60.34. mu.M. The IC50 of Peptide 5 is 159.6 mu M, the inhibition effect of the composite polypeptide on NB4 cells shows certain dose dependence, and the inhibition rate is increased along with the increase of the concentration of the polypeptide. The half inhibitory concentration of the composite polypeptide is obviously lower than that of the control polypeptide.

3) Effect of Complex Polypeptides on proliferation of hematological tumor cells

Artificially synthesizing a composite polypeptide on the basis of phage display screened polypeptides, respectively incubating tumor cells (K562 and NB 4), normal skin cells (HACAT) and skin cancer cells (A431) with artificially synthesized composite polypeptide Peptide 1 and polypeptide Peptide 5 with different concentrations (10 mu M, 1 mu M and 0.1 mu M) at 37 ℃ for 24h and 48h, detecting the influence of the composite polypeptide on tumor cell proliferation in 48h by adopting an MTT method, and adding a polypeptide group and a blank group without the polypeptide as negative controls. The results (see fig. 9) show that the complex polypeptide significantly inhibited the proliferative capacity of tumor cells (K562, NB 4) at a concentration of 10 μ M compared to the polypeptide Peptide 5 added group and the blank group without the polypeptide; but has no inhibition effect on normal skin cells (HACAT) and skin cancer cells (A431) and the composite polypeptide Peptide 1.

Reference to the literature

1. Gupta GP, Massague J: Platelets and metastasis revisited: a novel fatty link. J Clin Invest 2004, 114(12):1691-1693.

2.Gasic GJ, Gasic TB, Stewart CC: Antimetastatic effects associated with platelet reduction. Proc Natl Acad Sci U S A 1968, 61(1):46-52.

3.Nierodzik ML, Klepfish A, Karpatkin S: Role of platelets, thrombin, integrin IIb-IIIa, fibronectin and von Willebrand factor on tumor adhesion in vitro and metastasis in vivo. Thromb Haemost 1995, 74(1):282-290.

4.Nieswandt B, Hafner M, Echtenacher B, Mannel DN: Lysis of tumor cells by natural killer cells in mice is impeded by platelets. Cancer Res 1999, 59(6):1295-1300.

5.Jain S, Zuka M, Liu J, Russell S, Dent J, Guerrero JA, Forsyth J, Maruszak B, Gartner TK, Felding-Habermann B et al: Platelet glycoprotein Ib alpha supports experimental lung metastasis. Proc Natl Acad Sci U S A 2007, 104(21):9024-9028.

6.Placke T, Orgel M, Schaller M, Jung G, Rammensee HG, Kopp HG, Salih HR: Platelet-derived MHC class I confers a pseudonormal phenotype to cancer cells that subverts the antitumor reactivity of natural killer immune cells. Cancer Res 2012, 72(2):440-448.

7.Labelle M, Begum S, Hynes RO: Platelets guide the formation of early metastatic niches. Proc Natl Acad Sci U S A 2014, 111(30):E3053-3061.

8.Labelle M, Begum S, Hynes RO: Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. Cancer Cell 2011, 20(5):576-590.

9.Ibele GM, Kay NE, Johnson GJ, Jacob HS: Human platelets exert cytotoxic effects on tumor cells. Blood 1985, 65(5):1252-1255.

10.Ahmad R, Menezes J, Knafo L, Ahmad A: Activated human platelets express Fas-L and induce apoptosis in Fas-positive tumor cells. J Leukoc Biol 2001, 69(1):123-128.

11.Sagawa T, Tominaga A, Kodama T, Okada M: Cytotoxicity of unstimulated and thrombin-activated platelets to human tumour cells. Immunology 1993, 78(4):650-656.

12.Wang Y, Zhang H: Platelet-Induced Inhibition Of Tumor Cell Growth. Thromb Res 2008, 123:324-330.

13. Huyabin, Shao Liao, Zhao Lu, Shenying, Wukun, Wang Yiqiang, platelets activate AKT and ERK signaling pathways in leukemia cells L1210 and reduce their sensitivity to various drugs.Chinese experimental hematology magazine2016.

14.Echtler K, Konrad I, Lorenz M, Schneider S, Hofmaier S, Plenagl F, Stark K, Czermak T, Tirniceriu A, Eichhorn M et al: Platelet GPIIb supports initial pulmonary retention but inhibits subsequent proliferation of melanoma cells during hematogenic metastasis. PLoS One 2017, 12(3):e0172788.

15.Das B, Mondragon MO, Sadeghian M, Hatcher VB, Norin AJ: A novel ligand in lymphocyte-mediated cytotoxicity: expression of the beta subunit of H+ transporting ATP synthase on the surface of tumor cell lines. J Exp Med 1994, 180(1):273-281.

16.Dowling P, Meleady P, Dowd A, Henry M, Glynn S, Clynes M: Proteomic analysis of isolated membrane fractions from superinvasive cancer cells. Biochim Biophys Acta 2007, 1774(1):93-101.

17.Li W, Li Y, Li G, Zhou Z, Chang X, Xia Y, Dong X, Liu Z, Ren B, Liu W et al: Ectopic expression of the ATP synthase beta subunit on the membrane of PC-3M cells supports its potential role in prostate cancer metastasis. Int J Oncol 2017, 50(4):1312-1320.

18.Lu ZJ, Song QF, Jiang SS, Song Q, Wang W, Zhang GH, Kan B, Chen LJ, Yang JL, Luo F et al: Identification of ATP synthase beta subunit (ATPB) on the cell surface as a non-small cell lung cancer (NSCLC) associated antigen. BMC Cancer 2009, 9:16.

19.Ma Z, Cao M, Liu Y, He Y, Wang Y, Yang C, Wang W, Du Y, Zhou M, Gao F: Mitochondrial F1Fo-ATP synthase translocates to cell surface in hepatocytes and has high activity in tumor-like acidic and hypoxic environment. Acta Biochim Biophys Sin (Shanghai) 2010, 42(8):530-537.

20.Sun J, Yang ZL, Miao X, Zou Q, Li J, Liang L, Zeng G, Chen S: ATP5b and beta2-microglobulin are predictive markers for the prognosis of patients with gallbladder cancer. J Mol Histol 2015, 46(1):57-65.

21.Moser TL, Stack MS, Asplin I, Enghild JJ, Hojrup P, Everitt L, Hubchak S, Schnaper HW, Pizzo SV: Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc Natl Acad Sci U S A 1999, 96(6):2811-2816.

22.Veitonmaki N, Cao R, Wu LH, Moser TL, Li B, Pizzo SV, Zhivotovsky B, Cao Y: Endothelial cell surface ATP synthase-triggered caspase-apoptotic pathway is essential for k1-5-induced antiangiogenesis. Cancer Res 2004, 64(10):3679-3686.

23.Notari L, Arakaki N, Mueller D, Meier S, Amaral J, Becerra SP: Pigment epithelium-derived factor binds to cell-surface F(1)-ATP synthase. FEBS J 2010, 277(9):2192-2205.

24.Deshpande M, Notari L, Subramanian P, Notario V, Becerra SP: Inhibition of tumor cell surface ATP synthesis by pigment epithelium-derived factor: implications for antitumor activity. Int J Oncol 2012, 41(1):219-227.。

Sequence listing

<110> Suzhou university affiliated first hospital

<120> polypeptide targeting ectopic ATP5B pathway and application thereof

<141> 2018-03-29

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> chemical Synthesis (Artificial sequence)

<400> 1

Asn Pro Leu Lys Val Asp Trp Gly

1 5

<210> 2

<211> 8

<212> PRT

<213> chemical Synthesis (Artificial sequence)

<400> 2

Asn Pro Leu Lys Met Asp Trp Gly

1 5

<210> 3

<211> 10

<212> PRT

<213> chemical Synthesis (Artificial sequence)

<400> 3

Asn Pro Leu Lys Val Asp Trp Gly Leu Pro

1 5 10

<210> 4

<211> 10

<212> PRT

<213> chemical Synthesis (Artificial sequence)

<400> 4

Pro Val Asn Pro Leu Lys Val Asp Trp Gly

1 5 10

<210> 5

<211> 12

<212> PRT

<213> chemical Synthesis (Artificial sequence)

<400> 5

Pro Val Asn Pro Leu Lys Val Asp Trp Gly Leu Pro

1 5 10

<210> 6

<211> 10

<212> PRT

<213> chemical Synthesis (Artificial sequence)

<400> 6

Asn Pro Leu Lys Met Asp Trp Gly Leu Pro

1 5 10

<210> 7

<211> 10

<212> PRT

<213> chemical Synthesis (Artificial sequence)

<400> 7

Pro Val Asn Pro Leu Lys Met Asp Trp Gly

1 5 10

<210> 8

<211> 12

<212> PRT

<213> chemical Synthesis (Artificial sequence)

<400> 8

Pro Val Asn Pro Leu Lys Met Asp Trp Gly Leu Pro

1 5 10

Claims

1. A polypeptide targeting the ectopic ATP5B pathway, wherein the amino acid sequence of the polypeptide is NPLKDVDWG or the amino acid sequence shown in any one of SEQ ID NO.1-SEQ ID NO. 8.

2. Use of a polypeptide targeting the ectopic ATP5B pathway according to claim 1 in the preparation of a tumor diagnostic reagent.

3. Use of a polypeptide targeting the ectopic ATP5B pathway according to claim 1 for the preparation of a tumor-targeted medicament.

4. A tumor targeting therapeutic agent comprising the polypeptide of claim 1 which targets the ectopic ATP5B pathway.

5. The tumor targeted therapeutic agent of claim 4, wherein the polypeptide targeting the ectopic ATP5B pathway is conjugated to an active molecule comprising a toxic molecule having tumor treating effect.

6. The tumor targeted therapy agent according to claim 4, wherein the polypeptide targeting the ectopic ATP5B pathway is coupled to a drug delivery carrier, and the drug delivery carrier is a nanoparticle or a microsphere.

7. The tumor targeted therapy agent according to claim 4, wherein the polypeptide targeting the ectopic ATP5B pathway is expressed on a recombinant toxin molecule, and the recombinant toxin molecule is obtained by expressing a DNA sequence corresponding to the polypeptide targeting the ectopic ATP5B pathway and a DNA sequence of a protein toxin molecule through a recombinant gene after gene recombination.