CN116621928A - FITC-labeled CXCR4 binding peptide as well as preparation method and application thereof - Google Patents

Abstract

A fluorescent-labelled peptide, particularly preferably a peptide that binds to CXCR4, including peptides and fluorescent or dye molecules labelled to peptides; wherein the fluorescently labeled peptide exhibits one or more (at least two) of the following properties: binding to the extracellular loop of human CXCR4 (preferably the second extracellular loop) results in a KD of 2.5 nanomolar (nM) or less binding to human CXCR4 based on cell affinity assay testing. Such fluorescently labeled peptides can be used to identify CXCR4 positive tumor cells, as targeted therapeutic agents targeting the CXCR4 receptor.

Description

FITC-labeled CXCR4 binding peptide as well as preparation method and application thereof

Technical Field

The present application relates to peptides labeled with CXCR4 and/or other labels, particularly binding to CXCR4, and the use of such agents. The application also relates to methods of producing such labeled peptides. In particular to FITC (Fluorescein isothiocyanate ) labeled peptides, and a preparation method and application thereof, such as treatment of diseases involving CXCR4 expression.

Background

CXCR4 (CXC chemokine receptor 4) is a G-protein coupled receptor that modulates various physiological activities by binding to its cognate ligand CXCL12, also known as Stromal Cell Derived Factor-1 (SDF-1). CXCR4 plays an important role in mammalian development, mediating cell homing and migration processes, and plays a role in the early stages of embryoid cells. Both CXCR4 and SDF-1 knockout mice exhibit embryonic lethality, a substantially identical phenotype involving tissue and vascular abnormalities, supporting the hypothesis that CXCR4 is a key receptor for SDF-1 activity (CXCR 7 is the second receptor for SDF-1 is known). CXCR4 continues to be widely expressed in adults, with high levels of expression detected in bone marrow stem and progenitor cells, various circulating lymphocytes (B cells, activated T cells), endothelial precursor cells, tissue macrophages and fibroblasts.

The CXCR4-SDF-1 signaling axis plays an important role in chemotherapy-induced drug resistance and plays a key role in chemotherapy-induced Tumor Microenvironment (TME) by locally activating endothelial and myeloid progenitor cells, immune cell and progenitor cell infiltration, immunosuppression, and T cell activation. CXCR4 is overexpressed in a variety of human cancers, including breast, lung, colon, pancreas, brain, prostate, ovary, and hematopoietic (hematopoietic cancer). Clinical data indicate that CXCR4 overexpression is indicative of poor overall survival of hematological malignancies, breast cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, lung cancer, gynaecological cancer, liver cancer, prostate cancer and gall bladder cancer, and that some literature reports suggest that SDF-1 may act as a growth and/or survival factor for certain tumors through CXCR 4. In models of metastatic cancers, CXCR4 positive tumors have been demonstrated to metastasize to distant sites, and this activity is inhibited by drugs that silence the CXCR4 gene or antibodies that block CXCR4 or SDF-1. CXCR4 signaling pathways can also induce pro-angiogenic cytokines (such as VEGF), as well as integrins, adhesion molecules, and matrix degrading enzymes, which may mediate tumor cell invasion. Finally, CXCR4 expression was detected on tumor-infiltrating lymphocytes and fibroblasts, as well as tumor-associated macrophages, which tended to suppress immune recognition and attack the tumor, and remodel the tumor microenvironment to promote tumor growth and metastasis.

The multiple roles of CXCR4 in tumor growth, vascularization and metastasis, and its widespread expression in many common tumor types, make this receptor an attractive target for therapeutic intervention with inhibitory drugs, both peptide and small molecule inhibitors of CXCR4 have been identified and entered into the clinic, currently available products @Pliixafo) has been approved for mobilizing hematopoietic precursors in bone marrow for autologous stem cell transplantation.

The previous CXCR4 specific polypeptides or polypeptide-drug complexes (PDCs) were mainly MLB010 and MLB1701, etc. However, these peptides or PDCs are not labeled with fluorescence or isotopes. A staining or imaging agent capable of specifically recognizing cancer cells and tissues that overexpress CXCR4 would be an ideal drug for CXCR4 positive cancer targeted therapy.

Disclosure of Invention

The application aims to solve the problem that CXCR4 specific polypeptides or PDCs lack marks at present.

In a first aspect the present application provides a fluorescently labelled peptide, particularly preferably a peptide that binds to CXCR4, including peptides and fluorescent or dye molecules labelled onto the peptide; wherein the fluorescently labeled peptide exhibits one or more (at least two) of the following properties: binding to the extracellular loop of human CXCR4 (preferably the second extracellular loop) results in a KD of 2.5 nanomolar (nM) or less binding to human CXCR4 based on cell affinity assay testing.

In a preferred embodiment, the KD for binding to human CXCR4 is 1nM or less.

In a preferred embodiment, the peptide is an antibody or antigen binding fragment.

In a preferred embodiment, the peptide is derived from a peptide comprising, or preferably as shown belowSequence: ac-cycle [ Hcy-Tyr-Lys (iPr) - (D-Arg) -2Nal-Gly-Cys]-Lys(iPr)-PEG6-Lys-NH ₂ . It is reduced to the sequence shown in SEQ ID No. 1: hcy-Tyr-Lys-Arg-Xaa-Gly-Cys-Lys-Xaa-Lys.

In a preferred embodiment, the fluorescently labeled peptide comprises, or preferably is, the sequence shown below: ac-cycle [ Hcy-Tyr-Lys (iPr) - (D-Arg) -2Nal-Gly-Cys]-Lys(iPr)-PEG6-Lys(FITC)-NH ₂ 。

In a second aspect the present application provides a magnetic nanoparticle comprising a magnetic core material, a protein-based coating material coating the magnetic core material, and further comprising the fluorescently labeled peptide, the fluorescently labeled peptide being directly or indirectly coupled to the base coating material.

In a third aspect the present application provides a kit for detecting the presence or absence of circulating tumor cells expressing CXCR4, comprising a) coated magnetic nanoparticle comprising a magnetic core material, a protein-based coating material coating the magnetic core material, and further comprising said fluorescently labeled peptide, said fluorescently labeled peptide being directly or indirectly coupled to said base coating material; b) Cell specific dyes for excluding analysis of sample constituents other than tumor cells.

In a fourth aspect, the application provides a method of predicting the efficacy of a CXCR4 targeted therapy in a patient, or a method of detecting the presence of circulating tumor cells expressing CXCR4, or the use of a fluorescently labeled peptide, comprising:

a sample obtained by mixing the biological specimen with the fluorescent-labeled peptide;

the sample is analyzed to determine whether tumor cells expressing CXCR4 are present in the sample.

Wherein the analysis may be any one or more of multiparameter flow cytometry, immunofluorescent cytometry, laser scanning cytometry, bright field based image analysis, capillary volume analysis, spectral imaging analysis, manual cell analysis, and automated cell analysis.

In a preferred embodiment, the fluorescently labeled peptide is added in the form of the magnetic nanoparticle.

The method or application may be used for therapeutic/diagnostic purposes, or for non-therapeutic/diagnostic purposes (e.g., experimental verification during drug synthesis).

A method of synthesizing the fluorescently labeled peptide, comprising:

coupling Fmoc-protected amino acid and Fmoc-PEG6-OH from the C end one by a solid phase peptide synthesis method to form a raw material peptide, wherein at least Dde-protected Lys exists in the amino acid; removing Fmoc protection;

acetylation of the α -amino group; removing the Lys side chain protecting group Dde;

fluorescent labels are attached to the exposed side chains of the C-terminal Lys residues.

In a preferred embodiment, the Fmoc protected amino acid comprises: fmoc-Lys (iPr-Boc) -OH, fmoc-Cys (Trt) -OH, fmoc-Gly-OH, fmoc-2Nal-OH, fmoc-D-Arg (Pbf) -OH, fmoc-Lys (iPr-Boc) -OH, fmoc-Tyr (tBu) -OH and Fmoc-hCys (Trt) -OH.

In a preferred embodiment, the "fluorescent label attached to the exposed side chain of the C-terminal Lys residue" means: FITC in DIEA: DMF: dcm=12: 7:5, and reacting with the raw material peptide at the dark room temperature.

The application provides fluorescent CXCR4 binding peptides specifically binding to CXCR4, a synthetic method thereof, and applications such as detection of stained cells or tissues expressing CXCR 4. Such fluorescently labeled peptides can be used to identify CXCR4 positive tumor cells, as targeted therapeutic agents targeting the CXCR4 receptor, for diagnosis, imaging, or treatment.

Drawings

Fig. 1 is a dose response curve fit.

FIG. 2 is a flow cytometer analysis of FITC-labeled peptide binding.

FIG. 3 is an immunofluorescence image of a section of tumor tissue.

Detailed Description

The following description of the embodiments of the present application will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to fall within the scope of the application.

In the context of the present application, the meanings indicated by shorthand are as follows: ac: acetyl (Acetyl); boc: tert-butyloxycarbonyl (tert-butyloxycarbonyl); BOP: a catter condensing agent (benzotriazol-1-yloxy) -tris (dimethylamino) phosphonium hexafluorophosphate); bz: benzoyl (benzoyl); bzl/Bn: benzyl (benzyl); dab: diaminobutyric acid (1, 4-diaminobutyric acid); dap: diaminopropionic acid (2, 3-diaminopropionic acid); DCC: dicyclohexylcarbodiimide (dicyclohexyl-carbodiimide); DIC: and isopropyl carbodiimide (diisopropylcarbodiimide); DCM: dichloromethane (dichlormethane); DIEA: diisopropylethylamine (diisopropylethylamine); DMAP:4- (N, N-dimethylamino) pyridine (4- (N, N-dimethyl-amine) pyridine); DMF: n, N-dimethylformamide (N, N-dimethyl formamide); DMSO: dimethyl sulfoxide (dimethyl-sulfoxide); EDT:1, 2-ethanedithiol (1, 2-ethane-dithiol); et: an ethyl group; fmoc: 9-fluorenylmethoxycarbonyl (9-fluor-enylmethoxy carbonyl); HBTU: O-benzotriazole-N, N, N-tetramethylurea tetrafluoroborate (O-benzol-triazolyl-N, N, N ', N' -tetramethyluronium hexafluorophosphate); HOBt: hydroxybenzotriazole (hydroxybenzotriazole); hCys: homocysteine (homocysteine); iPr: isopropyl: IPA: isopropyl alcohol (isopropyl alcohol); me: a methyl group; mmt: 4-methoxytrityl (4-methoxytrityl); 2Nal: 2-naphthylalanine (2-naphthylalanine); 1Nal: 1-naphthylalanine (1-naphthylalanine); nle: norleucine (norleucine); NMM: n-methylmorpholine (N-methyl morpholine); NMP: n-methylpyrrolidone (N-methyl-pyrrosidone); orn: ornithine (orthinine); pbf:2, 4,6, 7-pentamethyl-dihydrobenzofuran-5-sulfonyl chloride (2, 4,6, 7-pentamethyl-dihydrobenzofurane-5-sulfonyl); PBS: phosphate buffer (phosphate buffered saline); pyBOP: benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate (benzotriazol-1-yloxy) -tris (pyrrolidino) -phosphonium hexafluoro-phosphate); pyBrOP: tripyrrolidinylphosphonium bromide hexafluorophosphate (bromotris (pyrrolidino) phosphonium hexafluorophosphate); tBu: a tertiary butyl group; TFA: trifluoroacetic acid (trifluoroacetic acid); TFE: trifluoroethanol (trifluoroethanol); THF: tetrahydrofuran (tetrahydrofuran); TIS: triisopropylsilane (triisopropyl silane); trt: trityl (trityl); all commonly used amino acids are designated by the military three letters unless specifically indicated.

Mass Spectrometry (MS) analysis: the preparation of the compounds in the following examples is an example, but is not intended to limit the scope of the application. In these examples, the observed molecular weight is reported as a deconvolution value (de-calculated value). Deconvolution values are derived from the formula MW (observations) =n (m/z) -n, where m/z represents the charge ion (positive charge pattern) and n is the number of charges of a particular substance. When multiple charged species are present in the mass spectrum, the observed molecular weight is reported as an average.

General methods of peptide synthesis, cyclic structure formation and salt exchange: peptides are synthesized using currently known solid phase peptide synthesis chemistry. The cyclic structure of these peptides is established by oxidation using air, or iodine, in the presence of an acidic acid, to form disulfide bonds.

Although the present application exemplifies the preparation of one particular peptide bond, other peptide bonds may be prepared at any time within the scope of the present application, for example, U.S. patent application Ser. No.15/695,862 filed on 5/9/2018, U.S. patent application Ser. No. 2018/0066021 published 16/6/2018 (U.S. patent publication No. US10,351,601 published 16/6/2019). In addition, other peptide bonds are within the scope of the present application and may be prepared by one of skill in the art who reads the U.S. patent application incorporated by reference herein.

Purification, salt form conversion and final product characterization: the final product was purified by reverse phase HPLC and further characterized by analytical HPLC and mass spectrometry. The polypeptide purified from reverse phase HPLC is typically in the form of trifluoroacetic acid (TFA). Such salts are typically converted to more pharmaceutically friendly salt forms, such as acetic acid or hydrochloride salt forms. In some cases, the end product is preferably acetate, hydrochloride, tartrate, or even citrate. Conversion of the peptide in the TFA salt to the hydrochloride salt may be accomplished by repeated lyophilization of the peptide in the TFA salt in a dilute hydrochloric acid solution. For converting peptides in TFA salts to acetate, the following procedure is generally used. Strong anion exchange resin (chloride form, substitution 3mmole/g, water content 50%, 2 g resin per gram of peptide) was first washed three times with milli Q water, then 3 times with 1N sodium hydroxide solution, 5 minutes/time, then 5 times with milli Q water, 5 minutes/time. The resin was washed with 75% ethanol water until a pH of around 7.4 was reached. The resin was treated with 10% acetic acid solution 3 times for 5 minutes each. The resin was then washed 3 times with 1% acetic acid solution for 5 minutes each. The resin can be used for salt conversion of purified peptides.

The purified lyophilized peptide was dissolved in 1% acetic acid solution and added to the resin prepared as described above. The mixture was stirred or magnetically stirred at room temperature for 1 hour. Separating the supernatant. The resin was washed three times with 1% acetic acid solution. The supernatant was combined with a wash solution, filtered through a 0.22 μm membrane and lyophilized to yield a polypeptide in acetate.

Example 1 synthesis of FITC-labeled CXCR4 binding peptide MB1831,

the MB1831 peptide chain is as follows:

Ac-cyclo[Hcy-Tyr-Lys(iPr)-(D-Arg)-2Nal-Gly-Cys]-Lys(iPr)-PEG6-Lys(FITC)-NH ₂

raw material peptide:

Fmoc-hCys (Trt) -Tyr (tBu) -Lys (iPr-Boc) - (D-Arg (Pbf)) -2Nal-Gly-Cys (Trt) -Lys (iPr-Boc) -PEG6-Lys (Dde) -AM Resin (Rink Amide Resin)

The starting peptides were assembled using standard Fmoc chemistry using RA resin (substitution capacity 0.4 mmom/g). Briefly, 1g of RA resin was swelled in 10mL of DCM for 10 min and then washed 4 times with dry DMF. Then two equivalents of Fmoc-Lys (Dde) -OH solution and five equivalents of DIEA solution were added to the resin. The resin mixture was stirred at room temperature for one hour. The reaction solution was drained from the resin bed and the resin was washed twice with DMF.

The resin was then added to a mixture of 10mL DCM/MeOH/DIEA (80:15:5, v/v) and shaken at room temperature for 10 min. The solution was drained from the resin and the resin was washed three times with DMF. To the resin was added 10mL of DMF in 25% piperidine and the mixture was shaken at room temperature for 20 minutes. This process was repeated once.

Fmoc protected amino acid residues were used: fmoc-Lys (iPr-Boc) -OH, fmoc-Cys (Trt) -OH, fmoc-Gly-OH, fmoc-2Nal-OH, fmoc-D-Arg (Pbf) -OH, fmoc-Lys (iPr-Boc) -OH, fmoc-Tyr (tBu) -OH and Fmoc-hCys (Trt) -OH were coupled according to the structure of the starting peptide, and the resin was washed before coupling the next amino acid: fmoc-PEG6-OH was activated by HOBt/DIC and 5DIEA was added. The coupling reaction was allowed to proceed for 1 to 1 half hour, and a negative ninhydrin test indicated that the reaction was complete. Fmoc was then removed with 10mL of 25% piperidine in DMF for 20 min.

After Fmoc-hCys (Trt) coupling of the last N-terminal residue. N-terminal Fmoc was removed using 20% piperidine in DMF and the acetylation of the α -amino group was performed with 22mL acetic anhydride/DIEA/DMF (1:1:4, v/v) at room temperature for 30min. Then the Lys side chain protecting group 1- (4, 4-dimethyl-2, 6-dioxapoxyhexidene) ethyl (Dde, 1- (4, 4-dimethyl-2, 6-dioxan-1-ylidene) -ethyl) was selectively removed with an aqueous solution of hydrazine in DMF (1:15, v/v) and washed 3 times for 5min each. After each DMF deprotection, the resin was washed (Bycroft, B.W.; chan, W.C.; chanabra, S.R.; hoc, N.D. chem. Commun.1993,778; diaz-Mochon, J.J.et al., org. Lett.2004, vol.6 (7), 1127-1129). Dde has been used as a protecting group for primary amines in the solid phase, typically cleaved under nucleophilic conditions using 2% v/v hydrazine.

In DIEA: DMF: dcm=12: 7:5 for 2.5 hours at room temperature in the absence of light, FITC (2 eq) was attached to the exposed side chain of the C-terminal Lys residue. After FITC coupling, the resin was washed with DMF and then DCM.

The resulting peptide was simultaneously deprotected and cleaved from the resin: use of 10mL TFA/EDT/titanium/H per gram resin ₂ O/thiazole/phenol (containing 81.5mL TFA,2.5Ml EDT,1.0mL,5.0mL water, 5.0mL of thiazole, and 5.0 grams of phenol per 100mL of solution) was reacted at room temperature for 70min. Then 8 volumes of methyl n-butyl ether were added to the cleavage mixture. The crude peptide precipitate was separated by centrifugation at 3000rpm for 3 min. The crude peptide precipitate was washed three times with methyl n-butyl ether. The crude peptide was then dissolved in aqueous acetonitrile and lyophilized.

The crude lyophilized product was used directly for cyclization reaction: the lyophilized crude peptide was dissolved in 0.5mg/mL (500 mg1 liter) of water and the pH of the solution was adjusted to 6.5 with magnetic stirring of a 1M ammonium carbonate solution. The hydrogen peroxide solution was added to the crude peptide solution with stirring to a final concentration of 0.03% to promote disulfide bond formation. Cyclization was completed within 1 hour. Cyclisation the final product was purified using reverse phase preparative column Daisogel (50X 250mm,8 m); mobile phase-solvent a:0.1% TFA in water; solvent B:0.1% TFA acetonitrile solution. Fractions containing the target product were combined and lyophilized (to give a TFA salt). Alternatively, the lyophilized crude linear peptide was redissolved in 5% acetonitrile/water (10:90, v/v) at a concentration of about 2mg/mL, and iodomethanol solution (3.6 g iodine in 500mL methanol) was added dropwise to the peptide solution until the reaction solution became pale yellow. A small amount of ascorbic acid solution (about 100 mg/mL) was added to the reaction mixture to reduce/neutralize additional iodine. The final cyclized peptide was purified by preparative HPLC and lyophilized to MB1831: ac-cycle [ Hcy-Tyr-Lys (iPr) - (D-Arg) -2Nal-Gly-Cys ] -Lys (iPr) -PEG6-Lys (FITC) -NH2.

Characterization of MB1831

Calculated mw2044.5da; MW2044.0Da was measured; analytical HPLC purity 97.4%; yellow lyophilized powder.

The agonist activity of MB1831 to inhibit the binding of SDF-1 to CXCR4 was detected by cell and nuclear receptor functional assays (Eurofin). In this cell-based assay system, SDF-1 induces cell signaling upon binding to CXCR4 and by monitoring Ca ²⁺ Activity (mobility) of the cells was examined for activity { Arakaki,1999#3432}. The IC50 value was half the maximum concentration of MB1831 concentration for SDF-1 inhibition.

The IC50 of MB1831 was 2.60nM as detected in the in vitro cell affinity assay; apparent dissociation constant (K) _B ) 0.11nM. Dose response curve fitting is shown in figure 1.

Example 2, flow cytometry identification of CXCR4 expressing cell lines

In this example, FITC-labeled CXCR4 binding peptide MB1831 was tested for binding to human and mouse cell lines by flow cytometry.

MB1831 (10 mM in ddH 20) was first diluted to a final concentration of 1. Mu.M with Phosphate Buffered Saline (PBS) buffer containing 3% BSA. Each cultured tumor cell line Daudi, MDA-MB-231, DU4475, U937, jurkat, MM-1S, NCIH520, SW480 and MCF-7 had 100 ten thousand cells each, washed twice with cold PBS, and then incubated with 100. Mu.L of 1. Mu.M MB1831 under ice or at Room Temperature (RT) for 2 hours. After incubation, cells were washed with PBS double and then resuspended with 500 μl cold PBS. FITC-labeled peptide binding was analyzed by flow cytometry using a FACScalibur flow cytometer. For each cell line, a threshold for positive staining was determined with control samples incubated with PBS. Using this threshold, less than 0.1% of the control cells were positive for FITC staining. Referring to FIG. 2, 93.1% of DAUDI cells, 30.1% of NCI-H520 cells, and 16.6% of SW480 cells were positive when incubated with MB 1831. The proportion of FITC positive cells in other cell lines was between 0.6% and 4.5%. The results show that the expression level of CXCR4 receptor in DAUDI cell line is highest and can be recognized by FITC-labeled CXCR4 peptide MB 1831.

Example 3 fluorescent immunohistochemistry of formalin-fixed Paraffin-embedded tumor tissue

MB1831 was dissolved in DMSO at a concentration of 1mg/ml and stored at-20 ℃. Formalin-fixed paraffin-embedded 4 μm-sized tumor tissue sections were removed. The sections were heated in a citric acid buffer (ph=6) at 95 ℃ for 30 minutes to obtain antigen activity, and then cooled at room temperature for 30 minutes. After three PBS washes, the slides were incubated with MB1831 for 1 hour at room temperature (1:2000 in PBS). The slides were then washed with PBS, counter-stained with 4', 6-diamine-2' -benzenedihydrochloride (DAPI; 1:1000; roche diagnostics Co.) and washed 3 times with PBS. The slide mounts a fluorescent medium to preserve the fluorescent signal.

Of the 20 breast cancer tumor tissue sections, 8 showed negative or weak staining for MB 1831. The 5 cases were moderately stained and the 7 cases were strongly stained, as shown in fig. 3 and table 1.

TABLE 1 detection results of 20 breast cancer tumor tissue sections

The above description of the specific embodiments of the application is given by way of example only, and the application is not limited to the specific embodiments described above. Any equivalent modifications and substitutions for the present application will occur to those skilled in the art, and are also within the scope of the present application. Accordingly, it is intended to cover such equivalent alterations and modifications as fall within the spirit and scope of the application.

Claims (10)

1. A fluorescently labeled peptide comprising a peptide and a fluorescent or dye molecule labeled onto the peptide; wherein the fluorescently labeled peptide exhibits one or more (at least two) of the following properties: the KD of binding to human CXCR4, as measured by cell affinity assay, is less than or equal to 2.5 nanomoles.

2. The fluorescently labeled peptide of claim 1, wherein said KD for binding to human CXCR4 is 1nM or less.

3. The fluorescently labeled peptide of claim 1, wherein said peptide is derived from a sequence comprising, or consisting of:

Ac-cyclo[Hcy-Tyr-Lys(iPr)-(D-Arg)-2Nal-Gly-Cys]-Lys(iPr)-PEG6-Lys-NH ₂ 。

4. the fluorescently labeled peptide according to claim 1, characterized in that it comprises, or preferably is, the sequence shown below:

Ac-cyclo[Hcy-Tyr-Lys(iPr)-(D-Arg)-2Nal-Gly-Cys]-Lys(iPr)-PEG6-Lys(FITC)-NH ₂ 。

5. a magnetic nanoparticle comprising a magnetic core material, a protein-based coating material encapsulating the magnetic core material, and further comprising the fluorescently labeled peptide of claim 1, the fluorescently labeled peptide being directly or indirectly coupled to the base coating material.

6. A kit for detecting the presence or absence of circulating tumor cells expressing CXCR4, comprising

a) Coated magnetic nanoparticles comprising a magnetic core material, a protein-based coating material coating the magnetic core material, and further comprising the fluorescently labeled peptide of claim 1, the fluorescently labeled peptide being directly or indirectly coupled to the base coating material;

b) Cell specific dyes for excluding analysis of sample constituents other than tumor cells.

7. A method of synthesizing the fluorescently labeled peptide, comprising:

coupling Fmoc-protected amino acid and Fmoc-PEG6-OH from the C end one by a solid phase peptide synthesis method to form a raw material peptide, wherein at least Dde-protected Lys exists in the amino acid; removing Fmoc protection;

acetylation of the α -amino group; removing the Lys side chain protecting group Dde;

fluorescent labels are attached to the exposed side chains of the C-terminal Lys residues.

8. The method of claim 7, wherein the Fmoc protected amino acid comprises: fmoc-Lys (iPr-Boc) -OH, fmoc-Cys (Trt) -OH, fmoc-Gly-OH, fmoc-2Nal-OH, fmoc-D-Arg (Pbf) -OH, fmoc-Lys (iPr-Boc) -OH, fmoc-Tyr (tBu) -OH and Fmoc-hCys (Trt) -OH.

9. The method of claim 7, wherein the "fluorescent label attached to the exposed side chain of the C-terminal Lys residue" is:

FITC in DIEA: DMF: dcm=12: 7:5, and reacting with the raw material peptide at the dark room temperature.

10. Use of a fluorescently labeled peptide according to claim 1, comprising:

a sample obtained by mixing the biological specimen with the fluorescent-labeled peptide;

the sample is analyzed to determine whether tumor cells expressing CXCR4 are present in the sample.