FAP-targeted polypeptide, molecular probe and application of FAP-targeted polypeptide

Abstract

The invention provides a polypeptide targeting FAP, a molecular probe and application of the polypeptide and the molecular probe, and belongs to the technical field of medical detection. The amino acid sequence of the polypeptide FAPW is shown as SEQ ID NO.1; the invention also provides a molecular probe for targeting fibroblast connection protein kinase FAP. The molecular probe is composed of the polypeptide FAPW and a signal unit connected with the polypeptide FAPW. The signal unit is a fluorescence label and / or a radioisotope label. The FAPW polypeptide provided by the invention has relatively strong binding capacity to FAP, and the KD value can reach 1.07 nM and is close to the affinity of an antibody. The molecular probe targeting FAP provided by the invention can be used for clinical common PET detection, is beneficial to later clinical transformation, can comprehensively display the FAP expression level of a tumor area and metastatic focus, and has good research prospects and clinical guidance significance.

Description

Technical Field

[0001] The present invention belongs to the field of medical detection technology, and in particular relates to a polypeptide targeting FAP, a molecular probe and applications thereof. Background Art

[0002] Cancer is a major global public health issue that poses a serious threat to human life and health. Early diagnosis and treatment of tumors are the key to improving their cure rates and the quality of life of patients. How to find and use effective clinical methods to achieve early diagnosis of tumors has become a hot area in basic research. The application of molecular imaging in the diagnosis and treatment of tumors has received increasing attention. The development of new high-affinity and specific molecular probes is an urgent problem to be solved in tumor molecular imaging. Past research on molecular probes for diagnosing tumors has mainly focused on screening specific markers expressed by tumor cells.

[0003] Fibroblast Activation Protein (FAP) is a highly conserved type II transmembrane serine protease, which is mainly expressed in activated fibroblasts and certain cancer-associated fibroblasts (CAFs). FAP is highly expressed in more than 90% of epithelial cancers, but almost not in normal tissues. However, it is significantly highly expressed in the tumor microenvironment of various solid tumors (such as breast cancer, lung cancer, colorectal cancer and pancreatic cancer). This selective expression makes FAP an important target for cancer diagnosis, treatment and prognosis assessment.

[0004] Probes targeting FAP have been a hot topic in tumor research in recent years, mainly including small molecule inhibitors, monoclonal antibodies, genetically engineered immune cells, and peptide-based targeted probes. Among them, targeted peptides have become an ideal targeting tool due to their good biocompatibility, high specificity, low immunogenicity, and easy modification. The application of targeted peptide probes has shown great potential in medical imaging, drug delivery, and tumor targeted therapy.

[0005] Although some probes targeting FAP have been developed for research or clinical practice, there are still some problems, such as low targeting efficiency, nonspecific binding to normal tissues, insufficient delivery efficiency, and poor in vivo stability. Therefore, designing and developing a FAP-targeting peptide probe with high efficiency, strong specificity and excellent pharmacokinetic properties has important scientific significance and clinical application value for early diagnosis, precise imaging and targeted treatment of tumors. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a polypeptide, a molecular probe and its application targeting FAP, and to explore its application potential in tumor imaging, targeted therapy, etc., so as to provide a new solution for the research and clinical application of FAP-related diseases.

[0007] The present invention provides a polypeptide FAPW targeting fibronectin kinase FAP, and the amino acid sequence of the polypeptide FAPW is shown in SEQ ID NO.1.

[0008] The present invention provides a nucleic acid molecule encoding the polypeptide FAPW.

[0009] The present invention provides biological materials containing the nucleic acid molecules, wherein the biological materials include recombinant DNA, expression cassettes, transposons, recombinant vectors, recombinant bacteria or recombinant cell lines.

[0010] The present invention provides a molecular probe targeting fibronectin kinase FAP, which is composed of the polypeptide FAPW and a signal unit connected thereto; the signal unit is fluorescently labeled and / or radioactive isotope labeled.

[0011] Preferably, the fluorescent label is one or more of IRDye800CW, Cy7, rhodamine and indocyanine green.

[0012] Preferably, the radioisotope label is 131 I. 177 Lu, 64 Cu, 99m Tc, 18 F and 68 One or more of Ga.

[0013] Preferably, the signal unit is connected to the polypeptide FAPW by chelation or click chemistry.

[0014] The present invention provides a composition for targeted treatment of diseases related to abnormal expression of fibronectin kinase FAP, comprising the polypeptide FAPW and an active drug for treating diseases related to abnormal expression of fibronectin kinase FAP.

[0015] Preferably, the diseases associated with abnormal expression of fibronectin kinase FAP include one or more of non-small cell lung cancer, melanoma, renal cancer, prostate cancer, colorectal cancer, pancreatic cancer, liver cancer, gastric cancer, esophageal cancer and breast cancer.

[0016] The present invention provides the use of the polypeptide FAPW and the molecular probe in preparing imaging preparations.

[0017] Compared with the prior art, the present invention has the following beneficial effects: a polypeptide FAPW targeting fibronectin kinase FAP, the amino acid sequence of the polypeptide FAPW is shown in SEQ ID NO.1, the FAPW polypeptide provided by the present invention has a strong binding ability to FAP, and the KD value can reach 1.07nM, which is close to the affinity of the antibody. The polypeptide FAPW can overcome the defects of monoclonal antibody molecular probes such as large molecular weight, easy inactivation, slow tissue penetration, and slow blood clearance while maintaining good targeting.

[0018] The molecular probe targeting fibronectin kinase FAP provided by the present invention is composed of the polypeptide FAPW and a signal unit connected thereto, can be used for PET detection commonly used in clinical practice, is conducive to later clinical transformation, can comprehensively display the FAP expression level in tumor areas and metastatic lesions, and has good research prospects and clinical guidance significance.

[0019] The preparation method of the molecular probe provided by the present invention is simple and easy, low in cost, highly practical, and has good biological safety and high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the molecular structural formula of the FAPW polypeptide of the present invention.

[0021] Figure 2 This is a mass spectrometry identification diagram of the FAPW polypeptide of the present invention.

[0022] Figure 3 This is the SPR affinity analysis of FAPW and FAP proteins in the present invention.

[0023] Figure 4 This is the confocal imaging of the FAPW polypeptide of the present invention in FAP-positive cells and negative cells.

[0024] Figure 5 Schematic diagram of the near-infrared fluorescence imaging results of the ICG-FAPW polypeptide probe in the present invention in living mice.

[0025] Figure 6 For the present invention 68 PET imaging of Ga-DOTA-FAPW mice. DETAILED DESCRIPTION

[0026] The present invention provides a polypeptide FAPW targeting fibronectin kinase FAP, and the amino acid sequence of the polypeptide FAPW is shown in SEQ ID NO.1, specifically as follows: TDKHEEDQYLC.

[0027] The present invention provides a nucleic acid molecule encoding the polypeptide FAPW, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2, specifically as follows: ACTGATAAACATGAAGAAGATCAATATTTATGT. The present invention does not specifically limit the source and preparation method of the polypeptide FAPW, and the conventional polypeptide preparation method in the art can be used. In the specific implementation of the present invention, the Fmoc solid phase peptide synthesis method can be used to prepare it. In the present invention, the amino acid residues of the polypeptide FAPW can be L-type, D-type, or a mixture of L- and D-types, as well as a sequence variation or a cyclic peptide structure in which the mirror image structure of the sequence is reversed.

[0028] The present invention also provides a biomaterial comprising the nucleic acid molecule, the biomaterial including but not limited to recombinant DNA, expression cassette, transposon, recombinant vector, recombinant bacteria or recombinant cell line. The present invention has no special limitation on the preparation method of the biomaterial, and the conventional preparation method in the art can be used.

[0029] The present invention provides a molecular probe targeting fibronectin kinase FAP, which is composed of the polypeptide FAPW and a signal unit connected thereto; the signal unit is fluorescently labeled and / or radioactive isotope labeled.

[0030] In the present invention, the fluorescent label is one or more of IRDye800CW, Cy7, rhodamine and indocyanine green; the radioisotope label is 131 I. 177 Lu, 64 Cu, 99m Tc, 18 F and 68 Ga. In the present invention, the signal unit is preferably connected to the polypeptide FAPW by chelation or click chemistry. In the present invention, the chelating agent is preferably DOTA, NOTA, DTPA or HYNIC; the click chemistry is labeled with NHS, EDC or MAL. The present invention has no special restrictions on the specific connection steps, and the methods and steps known in the art can be used.

[0031] In the present invention, indocyanine green ICG and radionuclide 68 Ga or 99m The dual-modal molecular probe obtained by Tc-labeled peptide FAPW has stronger selectivity and specificity, and can be detected by PET and near-infrared fluorescence imaging technology at the same time. Compared with other labeled peptide probes, it can better detect the expression of FAP.

[0032] The present invention provides a composition for targeted treatment of diseases related to abnormal expression of fibronectin kinase FAP, comprising the polypeptide FAPW and an active drug for treating diseases related to abnormal expression of fibronectin kinase FAP. In the present invention, the diseases related to abnormal expression of fibronectin kinase FAP include one or more of non-small cell lung cancer, melanoma, renal cancer, prostate cancer, colorectal cancer, pancreatic cancer, liver cancer, gastric cancer, esophageal cancer and breast cancer.

[0033] The present invention has no special limitation on the type of active drugs; including chemical drugs or biological drugs; or including nano drugs, radioactive drugs, photothermal therapy or photodynamic therapy drugs; further optional are alkylating agents, antimetabolites, anti-tumor natural drugs, anti-tumor antibiotics, anti-tumor angiogenesis drugs, metal complexes or tumor radiation targeting markers, etc.

[0034] In the present invention, the composition may optionally further include a drug carrier, which includes but is not limited to nanomaterials, liposomes and micelles.

[0035] The present invention also provides the use of the polypeptide FAPW and the molecular probe in the preparation of imaging preparations.

[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] Example 1

[0038] Preparation method of FAPW polypeptide targeting fibronectin kinase FAP

[0039] The amino acid sequence of the polypeptide FAPW is: TDKHEEDQYLC; the amino acid sequence is synthesized by solid phase synthesis using a standard Fmoc scheme. Finally, a deprotection process is performed: a DMF solution containing 20% ​​hexahydropyridine is used to remove the Fmoc group, the polypeptide is cleaved under the action of a strong acid (95% TFA, 5% H2O), and identified by mass spectrometry.

[0040] The molecular structure, mass spectrometry and liquid chromatography test results of FAPW are shown in Figures 1-2 As shown, it shows that the polypeptide has a high purity, proving the purity of the polypeptide and the correctness of the conjugate.

[0041] Example 2

[0042] Affinity analysis of peptide FAPW and FAP protein

[0043] In this example, the affinity between the polypeptide FAPW and the FAP protein is detected by the surface plasmon resonance (SPRi) method. The specific process is as follows:

[0044] 1 mg / mL peptide FAPW and 1×PBS buffer were spotted onto the chip and incubated overnight at 4°C in a humidified environment; then washed with 10×PBS for 10 min; then washed with 1×PBS for 10 min; finally washed twice with deionized water, 10 min each time;

[0045] The chip was immersed in 1×PBS containing 5% milk and incubated overnight at 4°C, then washed with 10×PBS for 10 min; washed with 1×PBS for 10 min; finally washed twice with deionized water, 10 min each time, dried with nitrogen, and loaded on the chip machine (Plexera HT surface plasmon resonance imaging system).

[0046] The mobile phase passed through 1×PBS, 2×PBS, 0.78μg / mL, 1.56μg / mL, 3.125μg / mL, 6.25μg / mL, 12.5μg / mL and 25μg / mL of human FAP purified protein in sequence, and the SPRi signal was recorded and analyzed.

[0047] The results are as follows Figure 3 As shown, it can be seen that the SPRi signal of FAPW gradually increases with the increase of protein concentration, indicating that the FAPW polypeptide prepared in Example 1 of the present invention has a strong binding ability to FAP, which can reach 1.07nM, close to the affinity of the antibody, and can be used for related application research.

[0048] Example 3

[0049] Confocal imaging to analyze affinity at the cellular level

[0050] This example detects the interaction between the polypeptide FAPW and the FAP high-expressing cells A549-FAP and the normal cells 293T, and the specific process is as follows:

[0051] After the last amino acid of the FAPW peptide was deprotected by solid phase synthesis, an excess of FITC was weighed and dissolved in a mixed solution of pyridine / DMF / DCM (12:7:5, v:v), and a small amount of DIEA (N,N-diisopropylethylamine) was added to adjust the pH to alkaline and react overnight in the dark. The synthesized peptide resin was added to 5 mL of lysis solution (95% TFA:2.5% Tis:2.5% H2O, v%), and the crude peptide was purified by HPLC after ether precipitation and vacuum drying and stored at -20°C. The non-small cell lung cancer cell line A549-FAP and normal human renal fibroblast 293T were cultured in DMEM culture medium containing 10% fetal bovine serum, and 1×10 5 / mL of cell concentration in a round glass-bottomed culture dish (35 mm), cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours, the culture medium was discarded, 1 μmol / L Hoechst 33342 and 10 μM FITC-labeled polypeptide were added to the two cells, respectively, incubated at 4°C in the dark for 20 minutes, and then washed three times with pre-cooled 1× PBS. The fluorescence distribution in the cells was detected using a laser scanning confocal microscope (ZEISS LSM 710).

[0052] The results are as follows Figure 4 As shown in the figure, after adding the FITC-labeled peptide FAPW, strong green fluorescence was observed on the A549-FAP cell membrane. The results show that the peptide FAPW binds to the cell membrane of positive cells, and the specificity is positively correlated with the expression level of the target protein. On the contrary, in the negative cell 293T, no green fluorescence signal was observed after adding the peptide, which further illustrates that the peptide FAPW specifically targets FAP.

[0053] Example 4

[0054] In vivo fluorescence imaging verifies the imaging performance of ICG-FAPW peptide

[0055] The FAP-positive cell line A549-FAP selected in the experiment was cultured in DMEM medium containing 10% FBS; BALB / C female 8-week-old mice were selected for the construction of tumor models. Each mouse was injected with 200 μL (2 μM) of ICG-labeled peptide FAPW (ICG-MAL was coupled with the thiol click reaction on the peptide FAPW) through the tail vein. After 30 minutes of injection, the mice were anesthetized with isoflurane and placed in the multi-spectral small animal in vivo imaging system IVIS Spectrum (PerkinElmer); the excitation wavelength was set to 805±10nm; the emission wavelength was 835±17.5nm, and the exposure time was 50ms for fluorescence detection. Finally, the mice were dissected, and the heart, liver, spleen, lung, kidney, and tumor were removed, and fluorescence imaging was performed under the same conditions as above.

[0056] like Figure 5 The results show that the prepared polypeptide imaging preparation ICG-FAPW was injected into tumor-bearing mice via the tail vein. The fluorescence signal of the tumor site gradually increased at 0.5h and reached a peak at 4h, proving that the molecular probe of the present invention has FAP targeting and can achieve high-sensitivity in vivo imaging of micro-tumors. The distribution of the probes in the main organs also shows that the polypeptide probe has good biocompatibility and safety.

[0057] Example 5

[0058] In vivo PET imaging of FAP peptide imaging agents

[0059] The peptide FAPW was commissioned to be synthesized by a biological company and DOTA was connected to the end.

[0060] 1 mg of the above-mentioned ICG-labeled peptide FAPW was dissolved in 1 mL of deionized water to obtain a peptide solution;

[0061] The germanium gallium (68Ge / 68Ga) generator (JSC Isotope) was eluted with 5mL of 0.1mol / L high-purity hydrochloric acid solution into an EP tube, 1mL of the solution with the highest radioactive content was collected, and 100μL of 1.25mol / L sodium acetate was added to adjust the pH value of the mixed solution to 3.5-4.5; 30μg of the polypeptide solution was added to the mixed solution and mixed thoroughly, and heated to 100°C for 10min; after the reaction was completed, the reaction solution was cooled to room temperature, and 4mL of sterile water for injection was added, and then filtered through a sterile filter membrane (0.22μm, 13mm) into a sterile product bottle to obtain a dual-modal molecular probe; 68 The radioactive labeling of Ga-DOTA-FAPW was performed using a dedicated radioactive detector for HPLC. The radioactive detection showed that its radiochemical purity was >99%. The FAP overexpressing cell line A549-FAP and the FAP underexpressing tumor A549 were quantified at 1×10 6 The cells were injected subcutaneously into the right hind limb of Balb / c mice. 3 In vivo imaging experiments were performed. 68 After the Ga-DOTA-FAPW probe solution was injected (the injection dose was 20 MBq, 150 μL), PET / CT (Discovery 790 Elite, GE healthcare) scanning imaging was performed for 15 min and 30 min under isoflurane-oxygen anesthesia.

[0062] The imaging results are as follows Figure 6 As shown, the uptake of FAPW by A549-FAP tumors was significantly higher than the background, but there was no obvious uptake of FAPW by A549 tumors. This indicates that the FAPW probe can specifically identify tumor models with high FAP expression, and can therefore be used as a PET probe to target FAP-expressing tumor imaging.

[0063] It can be seen from the above examples that the FAPW polypeptide provided by the present invention has a strong binding ability to FAP, which is close to the affinity of antibodies. The molecular probe composed of the polypeptide FAPW and the signal unit connected thereto can be used for PET detection commonly used in clinical practice, which is conducive to later clinical transformation, and can comprehensively display the FAP expression level in tumor areas and metastatic lesions, and has good research prospects and clinical guidance significance.

[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide FAPW targeting fibronectin kinase FAP, characterized in that: The amino acid sequence of the polypeptide FAPW is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the polypeptide FAPW according to claim 1.

3. A biological material comprising the nucleic acid molecule according to claim 2, characterized in that: The biological material includes recombinant DNA, expression cassette, transposon, recombinant vector, recombinant bacteria or recombinant cell line.

4. A molecular probe targeting fibronectin kinase FAP, characterized in that: It consists of the polypeptide FAPW according to claim 1 and a signal unit connected thereto; the signal unit is fluorescently labeled and / or radioactive isotope labeled.

5. The molecular probe according to claim 4, characterized in that The fluorescent label is one or more of IRDye800CW, Cy7, rhodamine and indocyanine green.

6. The molecular probe according to claim 4, characterized in that The radioisotope label is 131 I. 177 Lu, 64 Cu, 99m Tc, 18 F and 68 One or more of Ga.

7. The molecular probe according to claim 5 or 6, characterized in that The signal unit is connected to the polypeptide FAPW by means of chelation or click chemistry.

8. A composition for targeted treatment of diseases related to abnormal expression of fibronectin kinase FAP, characterized in that: The invention comprises the polypeptide FAPW according to claim 1 and an active drug for treating diseases related to abnormal expression of fibronectin kinase FAP.

9. The composition according to claim 8, characterized in that The diseases associated with abnormal expression of fibronectin kinase FAP include one or more of non-small cell lung cancer, melanoma, renal cancer, prostate cancer, colorectal cancer, pancreatic cancer, liver cancer, gastric cancer, esophageal cancer and breast cancer.

10. Use of the polypeptide FAPW according to claim 1 and the molecular probe according to any one of claims 4 to 7 in the preparation of imaging preparations.

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