阅读说明：本技术 一种整合素α6靶向多肽及其应用 (Integrin alpha 6 targeting polypeptide and application thereof ) 是由 曾木圣 冯国开 叶嘉聪 于 2021-08-27 设计创作，主要内容包括：本发明公开了一种整合素α6靶向多肽及其应用,其基序为RWYD,所述多肽具有更强的亲和力,平衡解离常数KD达到nM级别,亲和力获得极大提升,对整合素α6具有很强的亲和力,特异性强,无生物毒性,其在体内较为稳定且分布合理,可以作为靶向载体,整合素α6靶向多肽也可以作为分子探针应用于多种整合素α6高表达肿瘤的分子成像中。还提供了其二聚体,具有更显著的靶向作用,无生物毒性。整合素α6靶向多肽可应用于多种整合素α6高表达肿瘤的分子成像中在肿瘤分子成像中具有重要应用价值。(The motif of the integrin alpha 6 targeting polypeptide is RWDD, the polypeptide has stronger affinity, the equilibrium dissociation constant KD reaches the nM level, the affinity is greatly improved, the integrin alpha 6 has stronger affinity, the specificity is strong, the biotoxicity is avoided, the integrin alpha 6 targeting polypeptide is more stable and reasonable in distribution in vivo, and can be used as a targeting vector, and the integrin alpha 6 targeting polypeptide can also be used as a molecular probe to be applied to the molecular imaging of various integrin alpha 6 high-expression tumors. Also provides a dimer thereof, has more remarkable targeting effect and has no biological toxicity. The integrin alpha 6 targeted polypeptide can be applied to molecular imaging of various integrin alpha 6 high-expression tumors and has important application value in tumor molecular imaging.)

1. An integrin alpha 6-targeting polypeptide having the motif RWDD.

2. An integrin alpha 6-targeted polypeptide multimer polymerized from the polypeptide monomers of claim 1.

3. The multimer of claim 2, wherein the polypeptide has attached thereto a linker, preferably at least one of the following (I) to (II):

(I) a polyol;

preferably, the polyhydric alcohol is glycerol, pentaerythritol, xylitol, sorbitol, polyethylene glycol;

more preferably, the polyethylene glycol has a polymerization degree of 3-12;

(II) an amino acid;

preferably, the number of the amino acids is 3-8;

more preferably, the amino acid is lysine.

4. A multimer according to any of claims 2-3, wherein said multimer is a dimer.

5. Use of the targeting polypeptide of claim 1 or the multimer of any one of claims 2-4 for the preparation of the following targeted screening, diagnostic, therapeutic or prognostic assessment reagents.

6. A targeting agent comprising the polypeptide of claim 1 or the multimer of any of claims 2-4.

7. The agent according to claim 6, wherein the agent further comprises an imaging agent and/or a therapeutic agent.

8. The reagent of claim 7, wherein the imaging agent is at least one of an FB group, a radionuclide, biotin, a fluorophore, a fluorescent protein, an antibody, horseradish peroxidase, and alkaline phosphatase; preferably, the therapeutic agent is at least one of a radionuclide, a pro-apoptotic peptide, a nanoparticle, a chemotherapeutic agent, a nanodroplet, a liposomal drug, and a cytokine.

9. An agent according to claim 8, wherein the radionuclide is preferably a radionuclide labelled with the polypeptide of claim 1 or the multimer of any one of claims 2 to 4 by a chelating agent¹⁸F、⁹⁹Tc、⁶⁸Ga、⁶⁴Cu、¹¹¹In or¹⁷⁷Lu; preferably, the chelator is HYNIC, DOTA, NOTA or DTPA.

10. The reagent according to any one of claims 6 to 9, wherein the targeting agent is a tumor targeting agent, the tumor is a tumor with high integrin alpha 6 expression, and the tumor is preferably liver cancer, breast cancer, lung cancer, colorectal cancer, esophageal cancer, glioma, pancreatic cancer, prostate cancer, head and neck tumor, nasopharyngeal carcinoma, cervical carcinoma, gastric cancer, renal cancer, pheochromocytoma or paraganglioma.

Technical Field

The invention belongs to the technical field of targeting peptides, and particularly relates to an integrin alpha 6 targeting polypeptide and application thereof.

Background

The integrin is a membrane protein, has a large area outside a cell membrane, is beneficial to the approach and combination of molecular probes, and is an ideal target point for tumor molecular imaging and tumor targeted therapy. Integrins are composed of 18 alpha subunits and 8 beta subunits to form 24 heterodimers. Integrins overexpressed in tumors mainly include α v β 3, α v β 5, α 5 β 1, α 4 β 1, α 2 β 1, α 3 β 1, α v β 6, α 6 β 4 and α 6 β 1. Wherein, the RGD polypeptide molecular probe taking the integrin alphavbeta 3 as a target spot and the research of tumor targeted therapeutic drugs are the most extensive. The integrin alpha v beta 3 is over-expressed in tumor angiogenesis vessels and is low expressed in normal tissue vessels, so that the RGD polypeptide molecular probe can judge the condition of tumor angiogenesis. At present, researchers have successfully developed various RGD polypeptide radionuclide molecular probes, magnetic resonance molecular probes, ultrasonic molecular probes, and optical molecular probes. A considerable part of the RGD polypeptide molecular probes enter clinical experiments and achieve good imaging effect in tumor patients.

Integrin alpha 6 exists in vivo as α 6 β 4 and α 6 β 1 dimers, and integrin alpha 6 subunit is overexpressed in a variety of tumors, including breast cancer, liver cancer, lung cancer, colorectal cancer, esophageal cancer, glioma, pancreatic cancer, prostate cancer, head and neck tumors, nasopharyngeal carcinoma, cervical cancer, gastric cancer, renal cancer, pheochromocytoma, and paraganglioma. Integrin α 6 plays an important role in migration, invasion and proliferation of tumor cells. Meanwhile, the GEPIA database shows that the higher the integrin α 6 expression level, the worse the overall survival rate and disease-free survival rate of tumor patients. Therefore, integrin alpha 6 is an important marker for tumor diagnosis and prognosis evaluation, and the development of polypeptide diagnostic reagents using integrin alpha 6 as a target point has important significance for tumor diagnosis.

Disclosure of Invention

The invention aims to provide a novel integrin alpha 6 targeting polypeptide and application thereof.

The technical scheme adopted by the invention is as follows:

IN a first aspect of the invention, there is provided an integrin alpha 6-targeting polypeptide having the motif RWDD (SEQ IN NO: 1).

In some embodiments of the invention, the amino acids include L-form amino acids and D-form amino acids.

In a second aspect of the invention, an integrin alpha 6-targeting polypeptide multimer is provided, which is polymerized from a polypeptide monomer according to the first aspect of the invention.

In some embodiments of the invention, the polypeptide is linked to a linker.

In some preferred embodiments of the present invention, the linker is at least one of the following (I) to (II):

(I) a polyol;

(II) an amino acid.

In some embodiments of the invention, the polyol is glycerol, pentaerythritol, xylitol, sorbitol, polyethylene glycol.

In some preferred embodiments of the invention, the polyol is polyethylene glycol.

In some more preferred embodiments of the present invention, the polyol is polyethylene glycol having a degree of polymerization of 3 to 12.

In some embodiments of the invention, the number of amino acids is 3 to 8.

In some embodiments of the invention, the amino acid is any amino acid, preferably lysine.

In some embodiments of the invention, the linker is attached to the C-terminus of the polypeptide.

In some preferred embodiments of the invention, the multimer is a dimer.

In a third aspect of the invention, there is provided a polypeptide according to the first aspect of the invention or a multimer according to the second aspect of the invention for use in a targeted screening, diagnostic, therapeutic or prognostic assessment reagent.

In some embodiments of the invention, the targeting agent is a tumor targeting agent.

In some preferred embodiments of the invention, the tumor is a tumor with high expression of integrin α 6.

In some preferred embodiments of the invention, the tumor is preferably liver cancer, breast cancer, lung cancer, colorectal cancer, esophageal cancer, glioma, pancreatic cancer, prostate cancer, head and neck tumors, nasopharyngeal cancer, cervical cancer, gastric cancer, renal cancer, pheochromocytoma or paraganglioma.

In some embodiments of the invention, the application may also be for clinical intra-operative navigation NIRF zone optical imaging, Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET) or single photon emission imaging (SPET).

In a fourth aspect of the invention, there is provided a targeting agent comprising a polypeptide according to the first aspect of the invention or a multimer according to the second aspect of the invention.

In some embodiments of the invention, the targeting agent further comprises an imaging agent and/or a therapeutic agent.

In some preferred embodiments of the invention, the imaging agent is at least one of an FB group, a radionuclide, biotin, a fluorophore, a fluorescent protein, an antibody, horseradish peroxidase, and alkaline phosphatase.

In some more preferred embodiments of the invention, the imaging agent is a radionuclide.

In some embodiments of the invention, the nuclide is labelled with a chelator to the polypeptide of the first aspect of the invention or to the multimer of the second aspect of the invention.

In some preferred embodiments of the invention, the radionuclide is¹⁸F、⁹⁹Tc、⁶⁸Ga、⁶⁴Cu、¹¹¹In or¹⁷⁷Lu。

In some embodiments of the invention, the chelator is HYNIC, DOTA, NOTA or DTPA.

In some embodiments of the invention, the therapeutic agent is at least one of a radionuclide, a pro-apoptotic peptide, a nanoparticle, a chemotherapeutic agent, a nanodroplet, a liposomal drug, and a cytokine.

In some embodiments of the invention, the targeted agent is a drug.

In some embodiments of the present invention, the pharmaceutical formulation is an injection, a granule, an oral liquid, an aerosol, a capsule or a spray.

In some embodiments of the invention, the route of administration of the drug is intravenous, arterial, intracavity, subcutaneous, or intrathecal injection.

In some embodiments of the invention, the targeting agent is a tumor targeting agent.

In some preferred embodiments of the invention, the tumor is a tumor with high expression of integrin α 6.

In a more preferred embodiment of the invention, the tumor is liver cancer, breast cancer, lung cancer, colorectal cancer, esophageal cancer, glioma, pancreatic cancer, prostate cancer, head and neck cancer, nasopharyngeal cancer, cervical cancer, gastric cancer, renal cancer, pheochromocytoma or paraganglioma.

The invention has the beneficial effects that:

the invention provides a mutant RWDD, which has stronger affinity to integrin alpha 6, the equilibrium dissociation constant KD reaches the nM level, the affinity is greatly improved, the affinity to integrin alpha 6 is very strong, the specificity is strong, the mutant RWDD has no biotoxicity, the mutant RWDD is more stable and reasonably distributed in vivo, and can be used as a targeting vector, and integrin alpha 6 targeting polypeptide can be used as a molecular probe to be applied to the molecular imaging of various integrin alpha 6 high-expression tumors, such as navigation NIRF area optical imaging, Magnetic Resonance Imaging (MRI), positron emission imaging (positron emission tomography) PET, single photon emission imaging (SPET) and the like in clinical operation, and has important application value in the molecular imaging of tumors.

The invention also provides a dimer RD2 of the polypeptide, and the dimer RD2 is fully verified to have more obvious targeting effect on tumors with high integrin alpha 6 expression through a cell flow binding experiment, near infrared imaging in mice and PET/CT. Based on REWD and RD2, the invention also provides a targeting agent, which comprises RWDD or RD2, and also comprises an imaging agent and/or a therapeutic agent, so that the targeting agent has better tumor diagnosis and treatment effects, and the imaging agent marks corresponding polypeptide, so that the targeting agent has better stability and achieves better diagnosis effect.

Drawings

FIG. 1 is a structural formula of RWDD and its molecular imaging agents. Graph A is a structural formula of RWDD; panel B is a structural formula of RWDD-PEG 4-K-FITC; panel C is a structural formula of RWDD-PEG 4-K (ICG) -K (NOTA); diagram D is the structural formula of RWDD-PEG 4-K-NOTA; panel E is the structural formula of (RWDD-PEG 4) 2-K-K-FITC; f is the structural formula of (RWDD-PEG 4) 2-K-K-NOTA.

Fig. 2 is a graph of the strong affinity of RWYD for integrin α 6 β 4 or α 6 β 1. Panel a is the affinity of RWYD for integrin α 6 β 4; panel B shows the affinity of RWYD for α 6 β 1.

FIG. 3 shows the strong affinity of RWDD for tumor cells with high integrin α 6 expression. Panel A shows the expression of integrin α 6 in HCCLM3 cells; panel B shows the expression of integrin α 6 in Huh-7 cells; panel C shows the expression of integrin α 6 in Hep3B cells; d is the expression of integrin alpha 6 in S18 cells; panel E shows the expression of integrin α 6 in MDA-MB-231 cells; panel F shows the expression of integrin beta 4 in HCCLM3 cells; g picture is the expression of integrin beta 4 in Huh-7 cells; h picture is the expression of integrin beta 4 in Hep3B cells; panel I shows integrin beta 4 expression in S18 cells; panel J shows integrin beta 4 expression in MDA-MB-231 cells; k is the affinity of RWY, RD to HCCLM3 cells highly expressing integrin alpha 6; l plots affinity of RWY, RD to Huh7 cells highly expressing integrin α 6; m is the affinity of RWY, RD to Hep3B cells; n plots of affinity of RWY, RD to S18 cells highly expressing integrin α 6; o-plot affinity of RWY, RD to MDA-MB-231 cells highly expressing integrin alpha 6.

FIG. 4 shows the in vivo targeting of RYD to tumors that highly express integrin alpha 6. Panel A shows high uptake of RWDD-PEG 4-ICG in subcutaneous tumors of HCCLM3 that highly express integrin alpha 6; panel B shows that RWDD-PEG 4-ICG did not have high uptake in subcutaneous tumors of Hep3B that did not express integrin α 6; c is as shown in¹⁸Tumor uptake by the F-RWY marker; d is as follows¹⁸F-RD labeled tumor uptake.

FIG. 5 shows the strong affinity of RD, RD2 to tumor cells highly expressing integrin alpha 6. Panel A shows the affinity of RD, RD2 for HCCLM3 cells; panel B shows the affinity of RD, RD2 for S18 cells; panel C shows the affinity of RD, RD2 to HT29 cells; panel D shows the affinity of RD, RD2 to Huh7 cells; panel E shows the affinity of RD, RD2 for MDA-MB-231 cells; panel F shows the affinity of RD, RD2 to KYSE30 cells; g is the affinity of RD, RD2 to Hep3B cells; panel H shows the affinity of RD, RD2 for SW620 cells; panel I shows the affinity of RD, RD2 to SW1990 cells.

FIG. 6 shows the in vivo targeting of RD2 to tumors with high integrin alpha 6 expression. Panel A shows the affinity of RD for integrin α 6 β 4; panel B shows the affinity of RD2 for integrin α 6 β 4; panel C shows that in HCCLM3 subcutaneous liver cancer mouse,¹⁸F-RD2 uptake at the tumor; panel D is in HCCLM3 mouse model of liver cancer in situ,¹⁸F-RD uptake at the tumor; panel E shows that in HCCLM3 mouse model of liver cancer in situ,¹⁸F-RD2 uptake at the tumor.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

EXAMPLE 1 affinity assay of polypeptide mutants for integrin alpha 6

As a further improvement of the integrin alpha 6-targeting polypeptide, the amino acid motif is selected from the group consisting of: CRWYDENAC, CAWYDENAC, CRAYDENAC, CRWADENAC, CRWYAENAC, CRWYDANAC, CRWYDEAAC, RWYDENA, RWYDEN, RWYDANA, RWYDAN, RWYDEA, RWYDE, RWYDA, RWYD, RWYAEN, RWYAE and RWY. Wherein CRWYDENAC, CAWYDENAC, CRAYDENAC, CRWADENAC, CRWYAENAC, CRWYDANAC, CRWYDEAAC is a cyclic peptide formed by two terminal cysteines C, RWDDANA, RWDDENAN, RWDYANA, RWDDAN, RWDDEA, RWDDE, RWDYDE, RWDDA, RWDAY and RWY are linear peptides.

The affinity of the 18 polypeptides to integrin alpha 6 beta 4 was determined by a microcalorimetric electrophoresis (MST) method, which was performed as follows:

1) integrin α 6 β 4 protein was solubilized at 800nM and the polypeptide was solubilized at 100 μ M.

2) RED-tris-NTA secondary dye was diluted to 100nM using the MO series RED-tris-NTA protein labeling kit.

3) 100 mul alpha 6 beta 4 protein and 50 mul dye are mixed evenly and incubated for 30min at room temperature to combine the alpha 6 beta 4 protein and the dye.

4) Mu.l of the polypeptide was taken in a PCR tube and mixed with PBST (containing 0.05% Tween 20) at a ratio of 1: 1 dilution of the polypeptide to 16 concentration gradients.

5) 3 mul of protein/dye mixture is put into a PCR tube, and 3 mul of 16 polypeptides with different concentrations are respectively added and mixed evenly.

6) The mixed solution is sucked by a capillary tube and is detected on a machine (the model of the instrument is Monolith NT.115), and the parameters are set as 80 percent of LED/excitation power and medium MST power.

7) The equilibrium dissociation constant KD of α 6 β 4 protein to polypeptide was calculated by KD fitting mode using mo.

The results show that the linear 4-membered polypeptide RWDD (the structural formula is shown in FIG. 1A) has the strongest affinity with integrin alpha 6 beta 4 and is far higher than other 17 polypeptides, the KD of the linear 4-membered polypeptide RWDD is 21.82 + -3.86 nM (FIG. 2A), the KD is far higher than CRWYDENAC at 6.97 + -1.44 mu M, and the affinity is increased by 327 times relative to CRWYDENAC (RWY) (Table 1). Further, RWYD also had a strong affinity for integrin α 6 β 1 with a KD of 53.14 ± 28.69nM (fig. 2B).

TABLE 1 affinity assay of polypeptide mutants for integrin α 6 β 4

Example 2 affinity assay of mutant RWDD (RD) with tumor cells highly expressing integrin alpha 6

The expression conditions of integrin alpha 6 and beta 4 of human liver cancer cell HCCLM3, human liver cancer cell Huh-7, human liver cancer cell Hep3B, human nasopharyngeal carcinoma cell S18 and human breast cancer cell MDA-MB-231 are detected by flow type operation. The results indicated that integrin α 6 was expressed in HCCLM3, Huh-7, S18, and MDA-MB-231 cells (FIG. 3A, FIG. 3B, FIG. 3D, FIG. 3E); integrin beta 4 was expressed in HCCLM3, S18 and MDA-MB-231 cells (FIG. 3F, FIG. 3G, FIG. 3I, FIG. 3J); whereas Hep3B did not express α 6 and β 4 (fig. 3C, fig. 3H). Detecting the affinity of the tumor cells and the mutant RWDD by flow cytometry, and specifically operating as follows:

1) tumor cells are planted in a 12-hole plate, culture solution is added to the plate and placed in a cell culture box for 24 hours, so that the cells grow in an adherent manner, and each cell has 3 multiple holes.

2) Adding 2 μ l of RWDD-PEG 4-K-FITC (with structural formula shown in FIG. 1B, RD for short) or CRWYDENAC-PEG4-K-FITC (RWY for short) with concentration of 1mM into the cells, mixing, and incubating in an incubator at 37 deg.C for 2 hr.

3) The medium containing the polypeptide was aspirated off and the cells were washed 3 times with 500. mu.l/time of PBS.

4) Cells were trypsinized, medium resuspended, and centrifuged at 300g for 5 minutes.

5) The cell pellet was discarded from the supernatant, and 500. mu.l of physiological saline was resuspended in the cells and placed in a flow tube.

6) Detecting an FITC channel by a flow machine, and analyzing the result by CytExpert.

The results show that RD has a stronger affinity to tumor cells highly expressing integrin α 6 than RWY (fig. 3K, fig. 3L, fig. 3N, fig. 3O); while RD or RWY were consistent with the affinity of Hep3B cells that did not express integrin α 6 (fig. 3M).

Example 3 in vivo Targeted assay of RD and high integrin α 6 expressing tumors

In the small animal near infrared imaging, 2 μ l of fluorescent probe RWDD-PEG 4-K (ICG) -K (NOTA) (the structural formula is shown in figure 1C) with the concentration of 1mM is injected into HCCLM3 subcutaneous tumor-bearing mice through tail vein, and after circulating in vivo for 24 hours, the in vivo distribution of the subcutaneous tumor-bearing mice is observed. The results show that RWYD-PEG4-k (icg) -k (nota) was highly taken up in HCCLM3 subcutaneous tumors that highly expressed integrin α 6 (fig. 4A), but not in Hep3B subcutaneous tumors that did not express integrin α 6 (fig. 4B).

The tumor targeting of RD in HCCLM3 orthotopic liver cancer mouse is detected by PET/CT of small animal, and the specific operation is as follows:

1) 100 ug of RWDD-PEG 4-K-NOTA (the structural formula is shown in FIG. 1D), 6.275 ug of AlCl 3.6H₂0,330 μ l of absolute ethanol or acetonitrile, 5 μ l of glacial acetic acid, 65 μ l of 18F ionized water with an activity of about 3mCi, mixed until pH is about 4, placed in a 10ml vacuum glass bottle.

2) Heating in metal bath at 100 deg.C for 10min, and standing at room temperature for 5 min.

3) Waters Sep-Pak C18 column activation, 10ml absolute ethanol washing, after 20ml sterilization water washing.

4) 10ml of sterile water was added to the glass vial, mixed well, transferred to an activated C18 column and washed with 20ml of sterile water.

5) Eluting with 400 μ l of absolute ethanol, evaporating absolute ethanol with nitrogen blower, and dissolving with 300 μ l of normal saline.

6) HPLC check was performed using 10. mu. Ci for quality monitoring.

7) Approximately 100. mu. Ci was taken and injected into mice via tail vein.

8) After 1 hour, the mice were anesthetized with isoflurane and placed in a test chamber for PET/CT imaging.

The results show that it is possible to display,¹⁸f-labeled RWDD-PEG 4-K-NOTA (abbreviation)¹⁸F-RD) is clearly visible and is significantly better than¹⁸F-labeled CRWYDENAC-PEG4-K-NOTA (abbreviation)¹⁸F-RWY), except for a small uptake in the kidney and bladder, the background of other organs was low (fig. 4C, 4D). Both the results of small animal near-infrared imaging and PET/CT demonstrate in vivo that mutant RD has stronger targeting and specificity for integrin alpha 6 than RWY.

Example 4 affinity detection of dimer RWDD (RD 2 for short) and high integrin alpha 6-expressing tumor cells

RWDD-PEG 4 are linked by lysine (K) to form dimers or polyploids. The expression of the tumor cell integrin alpha 6 is detected by flow cytometry, and the result shows that HCCLM3, Huh-7, S18, MDA-MB-231, human colorectal adenocarcinoma cells SW620, human colon adenocarcinoma cells HT29, human esophageal squamous carcinoma cells KYSE30 and human pancreatic carcinoma cells SW1990 all highly express the integrin alpha 6, but Hep3B does not express the integrin alpha 6. The flow cytometry is used for detecting the affinity of the tumor cells to RD2, and the specific operation is similar to the operation of detecting the affinity of the tumor cells to RD by the flow cytometry. Results show that (RWDD-PEG 4)₂K-K-FITC (structural formula shown in figure 1E) has stronger affinity with tumor cells highly expressing integrin alpha 6 than RWDD-PEG 4-K-FITC (figure 5A-figure 5F, figure 5H-figure 5I); while RD2 or RD had similar affinity to Hep3B cells that did not express integrin α 6 (fig. 5G). This result demonstrates at the cellular level that RD can be increased by constructing dimer RD2 or polyploidsAffinity of syngen α 6.

Example 5 in vivo targeting assay of dimer RD2 with high integrin alpha 6 expressing tumors

Measured by the MST method (RWDD-PEG 4)₂-K-K-NOTA (structural formula shown in figure 1F) has stronger affinity with integrin alpha 6 beta 4, and K of the-K-NOTA_D64.83 + -28.16 nM (FIG. 6B), which is much greater than 642.38 + -260.52 nM of RWDD-PEG 4-K-NOTA (FIG. 6A), the increase in affinity is about 10-fold. The tumor targeting of RD2 in HCCLM3 subcutaneous liver cancer mice and in-situ liver cancer mice is detected by PET/CT of small animals, and the specific operation is similar to the detection of the tumor targeting of RD in HCCLM3 in-situ liver cancer mice. In HCCLM3 subcutaneous liver cancer mice,¹⁸f-labelled (RWDD-PEG 4)₂-K-K-NOTA (abbreviation)¹⁸F-RD2) was clearly visible at the tumor site uptake (fig. 6C). In the HCCLM3 orthotopic liver cancer mouse model,¹⁸the uptake effect of F-RD2 at the tumor is obviously better than that of F-RD2 at the tumor¹⁸F-RD, with a small uptake in the kidneys and bladder, had a lower background in other organs (fig. 6D, 6E). The result proves that the dimer RD2 has strong affinity with the integrin alpha 6 in a tumor-bearing mouse model, and has strong molecular targeting effect in tumors with high integrin alpha 6 expression.

The present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

SEQUENCE LISTING

<110> Zhongshan university, Zhongshan university tumor prevention and treatment center (Zhongshan university affiliated tumor hospital, Zhongshan university tumor research institute)

<120> integrin alpha 6 targeting polypeptide and application thereof

<130>

<160> 18

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