A novel VEGFR-3 targeting peptide and its screening and application

Abstract

The present invention relates to a novel VEGFR‑3 targeting peptide and its screening and application, and relates to the field of bioengineering technology. The targeting peptide of the present invention is TMVP1446 or TMVP1448; wherein the amino acid sequence of TMVP1446 is shown in SEQ ID NO: 1; the amino acid sequence of TMVP1448 is shown in SEQ ID NO: 2. The two polypeptides TMVP1446 and TMVP1448 screened by the screening method of the present invention have high affinity with VEGFR3 protein, can specifically target VEFGR‑3 protein both in vivo and in vitro, and the prepared imaging agent can be well applied to VEGFR‑3 molecular diagnosis of in situ tumors and metastatic tumors, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a novel VEGFR-3 targeting peptide and the screening and application thereof. Background Art

[0002] At present, the clinical evaluation of lymph node metastasis status mainly relies on preoperative imaging examinations (PET / CT, pelvic CT and MRI examinations) and postoperative pathological examinations. However, the sensitivity of CT in diagnosing cervical cancer lymph node metastasis is 42% (abdominal lymph nodes) and 79% (pelvic lymph nodes), and the sensitivity of PET-CT in diagnosing cervical cancer lymph node metastasis is only 50% (abdominal lymph nodes) and 83% (pelvic lymph nodes). The accuracy of existing preoperative examination methods cannot meet the needs of clinical diagnosis and treatment. Patients with early cervical cancer undergo pelvic lymph node dissection and lymph node pathological examination to confirm whether lymph node metastasis has occurred. However, pelvic lymph node dissection inevitably increases the scope of surgery and may cause many complications such as neurovascular injury, pelvic lymph cysts, pelvic tissue adhesions, infection, and local organ damage. The incidence of lymph node metastasis in patients with early cervical cancer is about 10%-20%, which indicates that more than 80% of patients undergo unnecessary lymph node dissection.

[0003] Therefore, the 2020 NCCN guidelines recommend that patients with stage I cervical cancer and lesions <2 cm can use sentinel lymph node imaging combined with ultramicroscopic pathology to assess lymph node metastasis. The sentinel lymph node examination strategy reduces surgical complications compared to conventional pelvic lymph node dissection, but the current sentinel lymph node (SLN) examination strategy requires fluorescent laparoscopes or radionuclide detection equipment, and ultramicroscopic pathology detection of lymph node metastasis requires high pathology costs. The sentinel lymph node imaging strategy itself also has the shortcomings of 20% unilateral or bilateral sentinel imaging failure, low intraoperative frozen section detection positive rate and false negatives. At present, there is an urgent need for a new technology or imaging agent to improve the sensitivity and specificity of the diagnosis of cervical cancer lymph node metastasis, so as to reduce unnecessary pelvic lymph node dissection in patients with early cervical cancer and achieve the goal of precision medicine.

[0004] The vascular endothelial growth factor C / D (VEGF-C / D) and vascular endothelial growth factor receptor-3 (VEGF R-3) signaling pathways are recognized as the most important lymphangiogenesis regulatory pathways to date. VEGFR-3 is mainly expressed in the embryonic period and in the newly formed lymphatic vessels induced by inflammatory tumors. Its expression gradually decreases with the maturation of lymphatic vessels, and it is not expressed in normal adults under non-pathological conditions. VEGFR-3 can be used as a monitoring reporter gene for the formation of new lymphatic vessels. VEGFR-3 is specifically expressed in newly formed lymphatic vessels, which enables it to dynamically monitor the formation of tumor newly formed lymphatic vessels, providing a reliable target for the development of new visualization imaging agents and therapeutic drugs for newly formed lymphatic vessels. In particular, multiple animal model studies using a visualized VEGFR-3-expressing mouse tumor model (VEGFR3 EGFPLUC) have confirmed that VEGFR-3 is only expressed in adult mice during the development of pathological conditions (including tumors and inflammation, etc.), and is expressed in large quantities during the development of tumor lymph node metastasis. The lymph nodes of normal adult mice in a healthy state do not express VEGFR-3.

[0005] Based on clinical needs and VEGFR-3 molecular expression characteristics, the present invention pre-screens VEGFR-3 molecular targeting peptides for application in VEGFR-3 molecular diagnosis of in situ tumors and metastatic tumors. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a novel VEGFR-3 targeting peptide and its screening and application, which can be applied to the molecular diagnosis of VEGFR-3 in situ tumors and metastatic tumors.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] The first object of the present invention is to provide a novel VEGFR-3 targeting peptide, wherein the targeting peptide is TMVP1446 or TMVP1448;

[0009] The amino acid sequence of TMVP1446 is shown in SEQ ID NO: 1;

[0010] Alternatively, the amino acid sequence of TMVP1448 is as shown in SEQ ID NO:2.

[0011] The second object of the present invention is to provide a novel method for screening VEGFR-3 targeting peptides, comprising the following steps:

[0012] (1) First round of screening: In vitro positive and negative screening of the bacterial flagellar peptide library against tumor metastasis-related VEGFR-3 protein and human IgG protein was performed to screen out peptides that bind to the VEGFR-3 protein;

[0013] First, the peptide library stock solution is taken for induction, and then human IgG protein and VEGFR-3 protein are added to the induced peptide library bacterial solution for binding, and finally the bacteria containing fusion proteins that can specifically bind to VEGFR-3 protein are screened out by using specific affinity.

[0014] (2) Second round of screening: The kinetic constants and affinity constants of the interaction between VEGFR-3 and the peptide were determined by SPR technology, and targeting peptides with high affinity to the VEGFR-3 protein were screened;

[0015] Using the Open SPRTM instrument, the carboxyl-modified chip was used as the SPR sensor chip, the carboxyl groups on the surface of the SPR chip were activated, and then the ligand protein VEGFR-3 was coupled to the chip. Finally, the kinetic constants and affinity constants of the interaction between VEGFR-3 and the polypeptide were measured to screen out targeting peptides with high affinity to the VEGFR-3 protein.

[0016] (3) The third round of screening: Establish an in vivo tumor-bearing model, screen the peptides with in vivo targeted distribution from the targeting peptides with high affinity to the VEGFR-3 protein, and finally obtain the targeting peptides that specifically target the VEGFR-3 protein.

[0017] An in vivo tumor-bearing mouse model was established. After labeling the peptides screened in the second round, the labeled peptides were injected into the model, and peptides with in vivo targeted distribution were screened from targeting peptides with high affinity to VEGFR-3 protein, and finally targeting peptides that specifically targeted VEGFR-3 protein were obtained.

[0018] Furthermore, the number of positive and negative screenings is 4 times.

[0019] The third object of the present invention is to provide an application of a novel VEGFR-3 targeting peptide, wherein the novel VEGFR-3 targeting peptide is used in the preparation of a visualization imaging agent.

[0020] The visualization developer contains a chelate, and the molecular formula of the chelate is 68 Ga-DOTA-CRR ALGGGTTSR or 68 Ga-DOTA-SLIGHGRGRRVR; 68 The molecular formula of Ga-DOTA-CRRALG GGTTSR is shown in formula (1), where 68 The molecular formula of Ga-DOTA-SLIGHGRGRRVR is shown in formula (2);

[0021]

[0022] DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid, which is chelated and connected with the VEGFR-3 targeting peptide and labeled with a radioactive nuclide tracer. 68 Ga.

[0023] Furthermore, the method for preparing the developer comprises the following steps:

[0024] (1) synthesizing peptides from DOTA and VEGFR-3 targeting peptides and preparing peptide solutions;

[0025] The chelating agent DOTA was chelated with the novel VEGFR-3 targeting peptide to synthesize the peptides DOTA-CRRALGGGTTSR and DOTA-SLIGHGRGRRVR, and the peptide solution was prepared.

[0026] (2) Rinse with hydrochloric acid solution 68 Ge / 68 Ga generator, collect eluent A, and detect 68 Radioactive dose of Ga;

[0027] (3) Mixing the sodium acetate solution and the eluent A evenly, and then mixing all of the mixture with the polypeptide solution to obtain a mixed solution, and heating the mixture to obtain a visualization developer.

[0028] Furthermore, the concentration of the polypeptide in the polypeptide solution is 0.8-1.2 mg / mL.

[0029] Furthermore, the concentration of the hydrochloric acid solution is 0.1-0.3M; the concentration of the sodium acetate solution is 1.20-1.30M.

[0030] Furthermore, the mixing volume ratio of the polypeptide solution, the hydrochloric acid solution and the sodium acetate solution is (0.009-0.011):(4-6):(0.090-0.095).

[0031] Furthermore, the heating reaction in step (3) is specifically: heating the reaction in a 100° C. metal bath for 15 minutes.

[0032] The beneficial effect of adopting the above further scheme is that the VEGFR-3 molecular targeting peptide screened by the present invention can be applied to the molecular diagnosis of VEGFR3 in in situ tumors and metastatic tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a verification of the tumor targeting of TMVP1446 in Example 1 of the present invention; wherein (A) is the specific imaging of 4T1 subcutaneous tumors in vivo; (B) is the SPR result diagram of TMVP1446 and human VEGFR3;

[0034] Figure 2 This is the verification of the tumor targeting of TMVP1448 in Example 1 of the present invention; wherein (A) is the specific imaging of 4T1 subcutaneous tumors in vivo; (B) is the SPR result diagram of TMVP1448 and human VEGFR3;

[0035] Figure 3 This is the in vivo distribution test of Cy7-TMVP1446 in 4T1 tumor-bearing mice in Example 2 of the present invention; wherein (A) is the fluorescence images of various organs and tumor tissues of 4T1 tumor-bearing mice at different time points; (B) is the fluorescence intensity statistics of various organs and tumor tissues of 4T1 tumor-bearing mice at different time points;

[0036] Figure 4 This is the in vivo distribution test of Cy7-TMVP1448 in 4T1 tumor-bearing mice in Example 2 of the present invention; wherein (A) is the fluorescence images of various organs and tumor tissues of 4T1 tumor-bearing mice at different time points; (B) is the fluorescence intensity statistics of various organs and tumor tissues of 4T1 tumor-bearing mice at different time points;

[0037] Figure 5 is the fluorescence signal intensity ratio of the tumor tissue to the control tissue muscle at each time point after the 4T1 tumor-bearing mice were injected with Cy7-TMVP1446 in Example 2 of the present invention;

[0038] Figure 6 is the fluorescence signal intensity ratio of the tumor tissue to the control tissue muscle at each time point after the 4T1 tumor-bearing mice were injected with Cy7-TMVP1448 in Example 2 of the present invention;

[0039] Figure 7This is an in vitro targeting test of TMVP1446 on VEGFR3 in Example 3 of the present invention; wherein (A) is a representative graph of the cell 5-TAMRA fluorescence signal of HeLaS3 and 4T1 cell lines VEGFR3 overexpression strains and control strains combined with 5-TAMRA-TMVP1446, and after competitive blocking treatment of the overexpression strains; (B) is a statistical graph of the average fluorescence intensity; (C) is a representative fluorescence image of the co-localization of 5-TAMRA-TMVP1446 and VEGFR3 taken up by HeLa S3 and 4T1 VEGFR3 overexpression cell lines; (D) is a representative fluorescence image of HeLa The in vitro uptake of 5-TAMRA-TMVP1446 by VEGFR3 overexpressing strains and control strains of S3 and 4T1 cell lines, as well as representative fluorescence images of the competitive blocking experimental group; (E) is the average fluorescence signal intensity of the 5-TAMRA fluorescence signal; (F) is a statistical graph of the average fluorescence signal intensity of the 5-TAMRA fluorescence signal; (G) is a representative fluorescence image of 5-TAMRA-TMVP1446 after being taken up by HUVEC and HLEC cell lines; in the figure, orange represents 5-TAMRA, blue represents DAPI-stained cell nuclei, and Merge represents the result of merging the red channel and green channel images;

[0040] Figure 8 This is an in vitro targeting test of TMVP1448 on VEGFR3 in Example 3 of the present invention; (A) is a representative graph of the fluorescence signal of 5-TAMRA of cells after the combination of 5-TAMRA-TMVP1448 and competitive blocking treatment of the OE strain in HeLaS3 and 4T1 cell lines overexpressing VEGFR3 (OE) and the control strain (CON); (B) is a statistical graph of the average fluorescence intensity; (C) is a representative fluorescence image of the co-localization of 5-TAMRA-TMVP1448 and VEGFR3 taken up by HeLaS3 and 4T1 VEGFR3 overexpressing cell lines; (D) is a representative fluorescence image of the HeLa The in vitro uptake of 5-TAMRA-TMVP1448 by VEGFR3 overexpressing strains (OE) and control strains (CON) of S3 and 4T1 cell lines, as well as representative fluorescence images of the competitive blocking experimental group; (E) is the average fluorescence signal intensity of the 5-TAMRA fluorescence signal; (F) is a statistical graph of the average fluorescence signal intensity of the 5-TAMRA fluorescence signal; (G) is a representative fluorescence image of 5-TAMRA-TMVP1448 after being taken up by HUVEC and HLEC cell lines; in the figure, orange is 5-TAMRA, blue is DAPI-stained cell nuclei, and Merge is the result of merging the red channel and the green channel;

[0041] Fig. 9This is an in vivo targeting test of TMVP1446 for VEGFR3 in Example 4 of the present invention; wherein (A) is a representative image of competitive blocking of TMVP1446 polypeptide and a representative image of mouse ex vivo organs and tumor tissues; (B) is a statistical result of signal-to-background ratio; (C) a representative image of subcutaneous tumor imaging 3 hours after Cy7-PEG4-TMVP1446 injection and a representative image of near-infrared fluorescence of mouse ex vivo subcutaneous tumors; (D) is a statistical result of signal-to-background ratio; (E) is a representative image of Cy7-PEG4-TMVP1446 imaging in A375 and A549 tumor models; (F) is a statistical result of signal-to-background ratio of Cy7-PEG4-TMVP 1446 imaging in A549 and A375 tumor models;

[0042] Fig.10 The in vivo targeting verification of TMVP1448 to VEGFR-3 of Example 4 of the present invention; wherein (A) the tumor targeting binding imaging results of Cy7-PEG4-TMVP1448 and free TMVP1448 and representative images of mouse ex vivo organs and tumor tissues; (B) the signal-to-background ratio of competitive blocking of free TMVP1448 polypeptide; (C) is a representative picture of subcutaneous tumor imaging 3h after injection of Cy7-PEG4-TMVP1448 and a representative near-infrared fluorescence image of mouse ex vivo subcutaneous tumor; (D) is the signal-to-background ratio 3h after injection of Cy7-PEG4-TMVP1448; (E) Cy7-PEG4-TMVP1448 imaging in A549 and A375 tumor models; (F) Cy7-PEG4-TMVP1448 signal-to-background ratio in A549 and A375 tumor models;

[0043] Fig.11 The imaging of sentinel lymph nodes of mouse tumors by TMVP1446 in Example 5 of the present invention; wherein (A) is a representative image of mouse SLN tracing; (B) is a picture of lymph nodes on the metastatic side of mouse SLN dissection; (C) is a representative near-infrared fluorescence image of mouse ex vivo SLN and contralateral normal lymph nodes;

[0044] Fig.12 The imaging of sentinel lymph nodes of mouse tumors by TMVP1448 in Example 5 of the present invention; wherein (A) is a representative image of mouse SLN tracing; (B) is a picture of lymph nodes on the metastatic side of mouse SLN dissection; (C) is a representative near-infrared fluorescence image of mouse ex vivo SLN and contralateral normal lymph nodes;

[0045] Fig.13 For Example 6 of the present invention 68Ga-DOTA-TMV1446 PET / CT imaging results of subcutaneous tumors; (A) is the ITLC detection result diagram; (B) is the representative imaging diagram at each time point; (C) is the dynamic change signal diagram of the local tumor;

[0046] Fig.14 For Example 6 of the present invention 68 Ga-DOTA-TMV1448 PET / CT imaging results of subcutaneous tumors; (A) is a representative imaging image at each time point in the mouse breast cancer model; (B) is a dynamic change signal image of the local tumor;

[0047] Fig.15 For Example 7 of the present invention 68 PET / CT images of metastatic lymph nodes by Ga-DOTA-TMVP1446;

[0048] Fig.16 For Example 7 of the present invention 68 Imaging of Ga-DOTA-TMVP1448 in the mouse tumor metastasis lymph node model; (A) is the imaging image in the mouse tumor lymph node metastasis model at 30 minutes and 60 minutes after administration; (B) is the imaging image in the mouse tumor lymph node metastasis model at 60 minutes after administration 18 F-FDG imaging images. DETAILED DESCRIPTION

[0049] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0050] Example 1: Screening of VEGFR-3 targeting peptides TMVP1446 and TMVP1448

[0051] (1) First round of screening: in vitro positive and negative screening of bacterial flagellar peptide library against the extracellular domain protein of tumor metastasis-related antigen VEGFR-3 and human IgG protein

[0052] ①Take a peptide library stock solution (Catalog no. K1125-01, Invitrogen, USA), melt it at room temperature and place it in a sterile conical flask, add 50 mL of IMC medium (containing 100 μg / mL ampicillin), and shake at 250 rpm at 25°C overnight for about 16 hours;

[0053] ②Take part of the overnight culture solution to measure OD 260 , according to 1OD 260 ≈1×109 cells, take 1×10 10 The bacterial solution of the cells was added to 50 mL of IMC medium (containing 100 μg / mL ampicillin), 500 μL of tryptophan was added, and the culture was shaken at 250 rpm and 25°C for 6 h to induce the expression of the bacterial flagellar peptide library to obtain the induced peptide library bacterial solution;

[0054] ③ Coat a 60 mm cell culture dish with 10 g / mL human IgG protein (1 mL ddH2O), rotate gently, and incubate at room temperature for 1 h;

[0055] ④ Add freshly prepared blocking solution, place on a horizontal shaker, and block at room temperature for 1 hour;

[0056] ⑤ When the peptide library induction is about to end, take a test tube, take out 10mL of the induced peptide library bacterial solution, add it to the upper tube, and vortex to mix;

[0057] ⑥ Remove the blocking solution from the IgG protein plate, add 10mL ddH2O to rinse gently, and discard; add the above peptide library solution, gently mix on a horizontal shaker at 50rpm for 1min, and keep at room temperature for 1h; take out another plate, coat with VEGFR-3 extracellular domain protein (Recombinant human VEGFR-3 / Flt-4 (Catal og no.349-F4)) 10μg / mL (1mL ddH2O), rotate gently, and keep at room temperature for 1h;

[0058] ⑦ After 1 hour, discard the blocking solution in the plate coated with the extracellular domain protein of VEGFR-3, aspirate the bacterial solution in the IgG protein plate (this is the peptide library bacterial solution that is not bound to the IgG protein) and add it, gently mix on a horizontal shaker at 50 rpm for 1 minute, and keep at room temperature for 1 hour;

[0059] ⑧ Discard the peptide library solution in the VEGFR-3 protein-coated plate (i.e., the peptide library solution that is not bound to the VEGFR-3 protein coating), wash gently for 5 minutes on a horizontal shaker at 50 rpm with 10 mL of washing solution, and repeat the washing for a total of 5 times to remove the peptide library solution that is not bound to the VEGFR-3 protein;

[0060] ⑨ After the last wash, remove the washing solution, place the plate on a vortex shaker for 30 seconds, add 10 mL of IMC medium (containing 100 μg / mL ampicillin) to rinse, aspirate the medium, place in a 50 mL centrifuge tube, shake at 250 rpm and 25°C overnight;

[0061] ⑩ Repeat the above positive and negative screening for a total of 4 times. After the last round of screening, dilute the overnight culture product of the shake bacteria in multiple proportions, apply it to RMG plates, and invert and culture at 25℃; dilute to 10 4The number of clones on the plate was large, the size was moderate, and it was easy to pick. 100 clones were randomly picked, shaken for amplification, and sent for sequencing after preservation.

[0062] The nucleotide sequence encoding the polypeptide in the sequencing results was translated into an amino acid sequence, and the polypeptide sequence was obtained by computer analysis. 54 polypeptides were obtained in the first round of screening.

[0063] (2) Second round of screening: Surface plasmon resonance (SPR) detection method was used to determine the kinetic constants (Ka, Kd) and affinity constant (KD) of the interaction between protein VEGFR-3 and peptides

[0064] ① Install the COOH chip as the SPR sensor chip on the OpenSPR instrument according to the Open SPRTM instrument standard operating procedure;

[0065] ② First, inject PBS buffer with pH = 7.4 at the maximum flow rate (150 μL / min) to calibrate the flow channel;

[0066] ③After the instrument reaches the signal baseline, load 200 μL of 80% isopropanol and continue running for 10 seconds to exhaust bubbles. After reaching the baseline, rinse the sample loop with PBS buffer and exhaust it with air;

[0067] ④After the signal reaches the baseline, adjust the buffer flow rate to 20 μL / min;

[0068] ⑤ Load 200 μL of EDC / NHS solution to activate the carboxyl chip, then rinse the sample loop with PBS buffer and evacuate with air;

[0069] ⑥ Load 200 μL of ligand protein VEGFR-3 (6 ug) diluted in activation buffer for chip coupling and run for 4 min;

[0070] ⑦ Take 200 μL of Blocking solution and load it; then rinse the sample loop with PBS buffer and empty it with air;

[0071] ⑧Observe the baseline for 5 minutes to ensure stability;

[0072] ⑨ Load the analyte solution with different concentrations, the binding time is 120s-240s; natural dissociation is 160s;

[0073] After the experiment, TraceDrawer (Ridgeview Instruments ab, Sweden) software was used to analyze and calculate the KD values ​​of the 52 peptides obtained in the first round of screening with VEGFR3 to evaluate the affinity of the peptides, where the analysis model was the One To One model.

[0074] Set 3×10 -5 M is the threshold, and peptides with KD values ​​less than this threshold are selected. In the second round of screening, 12 peptides with KD values ​​less than the set threshold of 3×10 -5 , these 12 peptides were renumbered and arranged from small to large according to KD values, and named TMVPX (X is 1446-1457).

[0075] (3) The third round of screening: in vivo tumor model screening

[0076] ① Fifty BALB / c Nude mice aged 4-6 weeks, weighing about 18 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Each mouse was subcutaneously inoculated with 4T1 tumor cells (1*10 4 After 2 weeks, the tumor tissue size was about 0.5-1 cm, and the breast cancer 4T1 subcutaneous tumor mouse model was established;

[0077] ② Entrust WuXi AppTec to synthesize the 12 peptides obtained in the second round of screening into fluorescent peptides coupled with the Cy7 group, named Cy7-PEG4-TMVPX (X is 1446-1457), and prepare Cy7-PEG4-TMVPX into 0.5 mg / mL fluorescent peptide solutions using sterile PBS;

[0078] ③ Each mouse was injected with 50uL of 0.5mg / mL fluorescent peptide solution through the tail vein, and 3 mice were injected with each fluorescent peptide solution. 3 hours after administration, the mice were subjected to small animal in vivo imaging scanning (PerkinElmer; USA) to detect the fluorescence signal intensity of the mice.

[0079] The results are as follows Figure 1-2 As shown:

[0080] (1) Figure 1 -B. Figure 2 -B, SPR detection showed that TMVP1446, TMVP1448 and VEGFR-3 protein had good affinity binding, and the equilibrium dissociation constant KD of the interaction between TMVP1446, TMVP1448 and VEGFR-3 protein was 1.93×10 -7 4.79×10 -7 ;

[0081] (2) Figure 1 -A. Figure 2 -A, after the 4T1 tumor-bearing mouse model was established, in the third round of screening, the tumor tissue could be clearly displayed 3h after intravenous injection of Cy7-TMVP1446 and Cy7-TMVP1448.

[0082] Example 2: Dynamic distribution of TMVP1446 and TMVP1448 molecular imaging probes in tumor-bearing mice

[0083] (1) Establishment of 4T1 breast cancer subcutaneous tumor mouse model:

[0084] Fifty 4-6 week old SPF BALB / c nude mice weighing about 18 g were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Each mouse was subcutaneously inoculated with 4T1 tumor cells (1*10 5 When the tumor volume reaches 300-500mm 3 The modeling is completed.

[0085] (2) In vivo mouse imaging experiments based on fluorescein and near-infrared probes

[0086] ① The mouse models prepared in (1) above were selected and randomly divided into two groups, namely the Cy7-PEG4-TMVP1446 group and the Cy7-PEG4-TMVP1448 group, with three mice in each group. 100 μL of 0.4 mM Cy7-PEG4-TMVP1446 solution or Cy7-PEG4-TMVP1448 solution prepared with sterile PBS was injected into the mice through the tail vein.

[0087] ② In the Cy7-PEG4-TMVP1446 group, mice were immediately killed and their hearts, livers, spleens, kidneys, lungs, intestinal tissues, and tumors were dissected and separated 1h, 3h, 8h, 24h, and 48h after the end of drug administration. The fluorescence signals of the organs were detected using a small animal imager. The detection mode was set to 840nm emission light, 745nm excitation light, automatic exposure, and Radiance efficiency (fluorescence) as the fluorescence signal display unit. The treatment process of the Cy7-PEG4-TMVP1448 group was the same as that of the Cy7-PEG4-TMVP1446 group, but the detection time points were set to 0.5h, 1h, 3h, 6h, and 24h.

[0088] ③The fluorescence detection signal was analyzed using LivingImage Version 4.0; using mouse muscle tissue as a control, the signal-to-background ratio (Tumor / Normal, T / N) of Cy7-PEG4-TMVP1446 and Cy7-PEG4-TMVP1448 in mice was calculated.

[0089] The experimental results are as follows Figure 3-6 As shown:

[0090] (1) Figure 3As shown in A and 3B, 3 hours after injection, the fluorescence signal of the whole body tissues of the mice in the Cy7-PEG4-TMVP1446 treatment group was the strongest. As time went on, the fluorescence signal of the whole body of the mice continued to decrease, and the signal intensity of the tumor and kidney was always higher than that of other organs and tissues. 1 hour after injection, the kidney signal was the strongest, the tumor signal was lower than that of the kidney, and 3 hours after injection, the tumor signal was higher than that of the kidney.

[0091] (2) Figure 4 As shown in A, the fluorescence signal of the whole body tissue of the mice in the Cy7-PEG4-TMVP1448 treatment group was the strongest 0.5h after injection, and the fluorescence signal of the whole body tissue of the mice decreased rapidly 0.5h-1h after injection. Figure 4 As shown in B; however, the signal intensity of tumor tissue was higher than that of other organ tissues at each time point.

[0092] (3) From Figure 5 , Figure 6 It can be seen that the signal-to-background ratio of Cy7-PEG4-TMVP1446 shows a trend of gradual increase with the extension of injection time, while the signal-to-background ratio of Cy7-PEG4-TMVP1448 gradually increases after injection, reaches a peak 6 hours after injection, and then begins to decline.

[0093] Example 3: Targeting of TMVP1446 and TMVP1448 to VEGFR3 in vitro

[0094] (1) Construction of tumor cell lines HeLa S3-VEGFR3 and 4T1-VEGFR3 that stably overexpress human VEGFR3

[0095] ① Lentivirus (overexpressing human VEGFR3 (NM-002020; commissioned Shanghai Gene Medical Technology Co., Ltd. to construct, amplify, and purify) was used to transfect HeLa S3 cells and 4T1 cells (ATCC),

[0096] ② HeLa S3 cells and 4T1 cells that had been transfected with lentivirus for 48 to 72 hours, were in good growth condition and about 70% confluent, and the medium was changed to screen and obtain tumor cell lines HeLa S3-VEGFR3(OE) and 4T1-VEGFR3(OE) that stably overexpressed human VEGFR3.

[0097] (2) Synthesis of 5-TAMRA-TMVP1446 and 5-TAMRA-TMVP1448

[0098] A peptide synthesis company was commissioned to synthesize 5-TAMRA-TMVP1446 and 5-TAMRA-TMVP1448 coupled with 5-hydroxy-tetramethylrhodamine (5-TAMRA) fluorescent group.

[0099] (3) Cell-binding peptide assay

[0100] ① Add HeLa S3-VEGFR3(OE)4T1-VEGFR3(OE) to be tested into the flow tube, with 5×10 4 cells; the cells were equally divided into six groups, namely 5-TAMRA-TMVP1446 (OE) group, 5-TAMRA-TMVP1446 (CON) group, 5-TAMR A-TMVP1448 (OE) group, 5-TAMRA-TMVP1448 (CON) group, and the other two groups were control groups (linear peptide blocking groups): 5-TAMRA-TMVP1446 (OE-blocking) group, 5-TA MRA-TMVP1446 (CON-blocking) group;

[0101] ② Add 0.1uM concentration of 5-TAMRA-TMVP1446 peptide solution, 5-TAMRA-TMVP1448 peptide solution, and 5uM concentration of linear peptide solution to each group of flow tubes; add them to each group of flow tubes respectively, mix well, and incubate on ice in the dark for 30 minutes; the flow tubes added with free linear peptide solution were incubated on ice for 5 minutes and tested on the machine.

[0102] (4) Flow cytometry to detect cell surface fluorescence intensity

[0103] ① The cells to be detected in (2) of this example were collected with trypsin and added to a flow cytometry tube, and then 100 μL of PBS containing 0.1% BSA was added.

[0104] ② According to the antibody instructions, add fluorescein isothiocyanate-labeled Anti-VEGF R3 antibody stock solution (PE anti-human VEGFR-3 (clone ID 9D9F9, BioLegend Catalog #356203) to the flow tube, and incubate the flow tube on ice for 20 minutes.

[0105] ③After the antibody incubation, the cells were detected by flow cytometry (Beckman Coulter, USA). Each experiment was repeated 3 times.

[0106] (5) Fluorescence colocalization

[0107] ① The co-incubated cells in (2) of this embodiment were inoculated into a 24-well plate and cultured in a cell culture incubator at 37°C. After the cells adhered to the wall, they were fixed with 4% paraformaldehyde (Wuhan Sewell Biotechnology Co., Ltd.), and VEGFR3 was labeled with an anti-VEGFR3 antibody labeled with fluorescein isothiocyanate, and finally DAPI (Wuhan Sewell Biotechnology Co., Ltd.) was added to stain the cell nucleus.

[0108] ② Observe the fluorescence intensity using an upright fluorescence microscope (Olympus, Japan). Figure 7 C, as shown in 8C.

[0109] (6) Cellular peptide uptake test

[0110] ① HeLa S3-VEGFR3 and 4T1-VEGFR3 were collected with trypsin, and the cells were inoculated into 24-well plates and cultured in a 37°C cell culture incubator (Heraeus Group, Germany).

[0111] ② After the cells adhered to the wall, the 24-well cell plate was taken out and the cells were divided into a blocking group (bloking) and an experimental group. The linear peptide was added to the bloking group and incubated in a 37°C incubator for 30 minutes.

[0112] ③ Take fresh sterile serum-free cell culture medium, prepare 10 μM concentration of 5-TAMRA-TMVP 1446 polypeptide solution, 5-TAMRA-TMVP1448 polypeptide solution and 100 μM concentration of TMVP1446 polypeptide solution, TMVP1446 polypeptide solution, respectively, aspirate 200 μL of the four polypeptide solutions, add them into the 24-well plates of HeLa S3-VEGFR3 cells and 24-well plates of 4T1-VEGFR3 cells respectively, put all the 24-well plates of cells back into the 37°C incubator and culture them in the dark for 2 hours.

[0113] ④Fix the cells with 4% paraformaldehyde, stain the cell nuclei with DAPI, and seal the sections with anti-fluorescence quenching sealing medium.

[0114] ⑤ Detect the fluorescence signal of 5-TAMRA under an upright fluorescence microscope (Olympus, Japan), and use ImageJ software to calculate its average fluorescence intensity.

[0115] (7) Cellular uptake in cell lines naturally expressing human VEGFR3

[0116] The experimental method was the same as that of Example (5), except that the cell line was replaced with human lymphatic endothelial cells (HLEC) naturally expressing human VEGFR 3 and human umbilical vein endothelial cells (HU VEC) as a negative control to evaluate the ability to take up 5-TAMRA-TMVP1446 and 5-TAMRA-TMVP1448.

[0117] The results are as follows Figure 7-8 As shown:

[0118] (1) In the cell binding peptide assay, Figure 7 A. Figure 8A shows that after incubation with 5-TAMRA-TMVP1446 and 5-TAMRA-TMVP1448, the fluorescence signals of HeLa S3-VEGF R3 (OE) group and 4T1-VEGFR3 (OE) group were significantly stronger than those of CON group;

[0119] (2) Figure 7 B. Figure 8 In B, for 5-TAMRA-TMVP1446 peptide and 5-TAMRA-TM VP1448 peptide, in HeLaS3 cell line and 4T1 cell line, the fluorescence intensity of OE strain was higher than that of CO N strain. 5-TAMRA-TMVP1446 has stronger specific binding ability to VEGFR3 highly expressed on the cell membrane than 5-TAMRA-TMVP1448, and the binding of 5-TAMRA-TMVP1446 and 5-TAMR A-TMVP1448 to cell lines highly expressing VEGFR3 can be specifically inhibited by free peptide;

[0120] (3) From Figure 7 C. Figure 8 As can be seen in F, in the cell uptake experiments of HeLa S3-VEGFR3 cells (OE) and 4T1-VEGFR3 cells (OE), 5-TAMRA-TMVP1446 and 5-TA MRA-TMVP1448 can specifically bind to VEGFR3 on the cell membrane;

[0121] (4) Figure 7 DG, Figure 8 In CG, the cell uptake experiment showed that 5-TAMRA-TMVP1446 and 5-TAMRA-TMVP1448 were specifically and highly taken up by the cell lines overexpressing VEGFR-3, and the uptake could be blocked by the corresponding free linear peptides; and the naturally overexpressed VEGFR-3 cell line HLEC also highly took up 5-TAMRA-TMVP1446 and 5-TAMRA-TMVP1448.

[0122] Example 4: In vivo targeting test of TMVP1446 and TMVP1448 for VEGFR3

[0123] (1) In vivo competition blocking assay

[0124] ① Establish a 4T1 subcutaneous tumor-bearing mouse model using the modeling method of Example 2;

[0125] ② Select 6 mice bearing subcutaneous breast cancer 4T1 tumors prepared in (1) above and randomly divide them into two groups: a competitive blocking group (Blocking) and a control group (Control), with 3 mice in each group. Inject 100uL, 0.1uM Cy7-PEG4-TMVP1446 solution and Cy7-PEG4-TMVP1448 solution into the mice in the Control group through the tail vein. First, inject 100uL, 5uM TMVP1446 solution and TMVP1448 solution into the mice in the Blocking group, respectively, and then inject 100uL, 0.1uM Cy7-PEG4-TMVP1446 solution and Cy7-PEG4-TMVP1448 solution;

[0126] ③ After the administration, the whole body of the mouse was subjected to fluorescence detection using a mouse live imaging instrument, and then the heart, liver, spleen, kidney, lung, intestinal tissue, and tumor of the mouse were dissected and separated, and the fluorescence signal was detected to calculate T / N. The detection mode and analysis and calculation method were the same as those in Example 2.

[0127] (2) Imaging of Cy7-PEG4-TMVP1446 and Cy7-PEG4-TMVP1448 in HeLa S3 cell lines with differentially expressed VEGFR3 tumors

[0128] ① Establishment of a subcutaneous tumor mouse model: The VEGFR3-overexpressing cell line (OE) and the control cell line (CON) of the HeLa S3 cell line established in Example 3 were selected, and a mouse subcutaneous tumor model was established by the modeling method in Example 2.

[0129] ② Imaging test: 100 μL and 0.1 μmoL of Cy7-PEG4-TMVP1446 and Cy7-PEG4-TMVP1448 were injected into the mouse model through the tail vein, and the whole body of the mouse was fluorescently detected. The mouse injected with Cy7-PEG4-TMVP1448 was scanned and imaged 3 hours after injection; the imaging time point of Cy7-PEG4-TMVP1448 was 1 hour after injection; then the tumor of the mouse was dissected and separated, and its fluorescence signal was detected, and T / N was calculated. The detection mode, analysis and calculation method are the same as those in Example 2.

[0130] (4) Imaging tests in other tumors

[0131] A375 cell line and A549 cell line were selected to establish subcutaneous xenograft tumor models of human melanoma A375 and human lung cancer A549 respectively by the modeling method in Example 2; the number of cells required for the A375 cell line was 5×10 5 The number of cells required for A549 cell line is 3×10 6 Piece / piece.

[0132] The imaging test method of (2) in this embodiment was used to perform imaging tests on subcutaneous xenograft tumor models of human melanoma A375 and human lung cancer A549.

[0133] The results are as follows Figure 9-10 As shown:

[0134] (1) By Fig. 9 AB, Fig.10 AB showed that the fluorescence signal of the subcutaneous tumor in mice was significantly weakened after competitive blocking, and the fluorescence signal of the tumor tissue was lower than that of the control group. The T / N values ​​of Cy7-PEG4-TMVP1446 and Cy7-PEG4-TMVP1448 also decreased compared with the control group.

[0135] (2) Fig. 9 CD, Fig.10 As shown in CD, for Cy7-PEG4-TMVP1446 and Cy7-PEG4-TMVP1448, specific near-infrared fluorescence signals could be detected locally in the tumors of both CON and OE group mice.

[0136] (3) By Fig. 9 EF, Fig.10 EF, in human melanoma A375 and human lung cancer 549 models, Cy7-PEG4-TMVP1446 and Cy7-PEG4-TMVP1448 can clearly show the tumor localization with high signal-to-background ratio.

[0137] Example 5: Imaging of sentinel lymph nodes of mouse tumors by TMVP1446 and TMVP1448

[0138] (1) Establishment of mouse lymph node metastasis model

[0139] Fifty SPF-grade BALB / c nude mice aged 4-6 weeks, weighing approximately 18 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The paw pads of each mouse were inoculated with 4T1 tumor cells (2.5*10 4 The mouse tumor lymph node metastasis model was established.

[0140] (2) Fluorescence signal detection

[0141] On the 9th day after the establishment of the mouse lymph node metastasis model, the mice were randomly divided into two groups, Cy7-PEG4-TMVP1446 group and Cy7-PEG4-TMVP1448 group; 100uL of 0.1uM Cy7-PEG4-TMVP1446 peptide solution and Cy7-PEG4-TMVP1448 peptide solution were injected through the tail vein, respectively.

[0142] After the mice were anesthetized, the whole-body fluorescence signals of the mice were detected using a small animal in vivo optical imager (PerkinElmer, USA). The tumor sentinel lymph nodes and contralateral normal lymph nodes of the mice were then dissected and separated, and their fluorescence signals were detected. The imaging time of the two groups of mice was the same as that of the in situ tumor model in Example 2.

[0143] The results are as follows Figure 11-12 We can get:

[0144] (1) Fig.11 A. Fig.12 As shown in A, the blue on the left side of the mouse is the normal lymph node, and the red on the right side is the tumor SLN. Near-infrared fluorescence signals can be detected at the SLN position in both the Cy7-PEG4-TMVP1446 group and the Cy7-PEG4-TMVP1448 group ( Fig.11 B. Fig.12 B).

[0145] (2) Fig.11 As shown in C and 12C, Cy7-PEG4-TMVP1446 and Cy7-PEG4-TMVP 1448 both have better specific tracing effects on SLN.

[0146] Example 6: Synthesis of imaging agents and their application in tumor imaging experiments

[0147] (1) Imaging agent 68 Ga-DOTA-TMVP1446, 68 Synthesis of Ga-DOTA-TMVP1448

[0148] ①Use ultrapure water to prepare a peptide solution with a concentration of 1 mg / mL from the peptide freeze-dried powder connected to the chelating agent DOTA, and divide it into 50 μL EP tubes and freeze it in a -20°C refrigerator.

[0149] ②Take 5mL of 0.1M HCl solution and slowly rinse 68 Ge / 68 Ga generator, and use 1.5mL EP tubes to collect eluent A, and use a radiation dose meter to detect the radioactive dose in each tube.

[0150] ③ Take 93 μL of 1.25M sodium acetate (NaAc) solution and mix it with 1 mL of eluent A, then transfer the mixed solution to a clean EP tube containing 10 μL of polypeptide solution.

[0151] ④ Mix all the liquid in the EP tube, place it in a 100℃ metal bath and heat it for 15 minutes. After the reaction is completed, take out the EP tube and let it cool naturally to room temperature to obtain the marked liquid.

[0152] ⑤ Pass the C18 column with 10 mL of anhydrous ethanol and 10 mL of ultrapure water in sequence, activate the C18 column in advance, carefully pass the labeled liquid cooled to room temperature through the C18 column, then elute the residual liquid with 10 mL of ultrapure water, and then rinse the C18 column with 0.5 mL of 75% ethanol, and collect the eluent B.

[0153] ⑥ Dilute eluent B to 4 mL with sterile PBS solution. After the ethanol concentration in eluent B is less than 10%, sterilize the diluted eluent and store it in a sterile container. 68 Ga-DOTA-TMV1446 was purified by Sep-Pak C18 Light column and then analyzed by ITLC. Fig.13 As shown in A.

[0154] (2) Establishment of a mouse 4T1 breast cancer subcutaneous tumor model

[0155] Four-week-old BALB / c mice were used and the modeling method of Example 2 was used to establish a mouse 4T1 breast cancer subcutaneous tumor model.

[0156] (3) PET / CT mouse scanning imaging

[0157] ① Six mice with subcutaneous tumor models established in this example were randomly divided into two groups, with 3 mice in each group, and 200±10μCi of labeled 68 Ga-DOTA-TMV1446, 200±10μCi labeled 68 Ga-DOTA-TMV1448, the peptide volume was controlled below 100 μL;

[0158] ② After the imaging agent was injected into the tail vein, the whole body of the mouse was continuously scanned and images were collected at 30s, 5min, 20min, 40min, and 60min. The results are as follows Fig.13 B. Fig.13 C. Fig.14 A, as shown in 14B.

[0159] Depend on Figure 13-14 As shown:

[0160] (1) 68 Ga-DOTA-TMV1448 can show subcutaneous tumors, Fig.13 A is the radiochemical purity diagram detected by ITLC-SG. 68 The radiochemical purity of Ga-DOTA-TMVP1446 is greater than 95%; Fig.13 B Representative images of breast cancer in mice at different time points, with the top image being the coronal image. The white circle in the image indicates the tumor site. Fig.13 A broken line in C represents a mouse.

[0161] (2) By Fig.14 Available 68 Ga-DOTA-TMV1448 can show subcutaneous tumors, Fig.14 B is 68 Dynamic changes of Ga-DOTA-TMVP1448 signal in local tumors.

[0162] Example 7: Application of TMVP1446 and TMVP1448 in imaging of tumor metastatic lymph nodes in mice

[0163] ① The modeling method of Example 5 was used to establish a lymph node metastasis model mouse, and 6 lymph node metastasis model mice were randomly selected and randomly divided into two groups, namely 68 Ga-DOTA-TMV1446 group, 68 Ga-DOTA-TMV1448 group, 3 mice in each group;

[0164] ② On the 9th day after the model was established, the mice were anesthetized and 68 Ga-DOTA-TMV1446 group mice, 68 The mice in the Ga-DOTA-TMV1448 group were injected with 200±10μCi of labeled 68 Ga-DOT A-TMV1446, 68 Ga-DOTA-TMV1448, the peptide volume was controlled below 100 μL;

[0165] ③ 68 The Ga-DOTA-TMV1446 group was scanned after the imaging agent was injected into the tail vein, first with PET and then with CT; 68 The Ga-DOTA-TMV1448 group underwent PET / CT scanning using a small animal imaging device (Novel Medical, Beijing, China) at 30 and 60 minutes after administration. The same mice were injected with the tail vein 24 hours later. 18 F-FDG 100 μCi, and PET / CT scanning was performed 60 min after administration.

[0166] The results are as follows Figure 15-16 As shown:

[0167] 68 Ga-DOTA-TMVP1446, 68 Ga-DOTA-TMVP1448 can display metastatic lymph nodes, and the imaging effect is correlated with metastasis.

[0168] In summary, the two polypeptides TMVP1446 and TM VP1448 screened by the screening method of the present invention have high affinity with VEGFR3 protein and can specifically target VEFGR-3 protein both in vivo and in vitro; the prepared imaging agent can be well applied to molecular diagnosis of VEGFR-3 in situ tumors and metastatic tumors. Therefore, TMVP1446 and TMVP1448 have good application prospects.

[0169] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A VEGFR-3 targeting peptide, characterized in that: The targeting peptide is TMVP1446 or TMVP1448; The amino acid sequence of TMVP1446 is shown in SEQ ID NO: 1; Alternatively, the amino acid sequence of TMVP1448 is as shown in SEQ ID NO:

2.

2. An application of a VEGFR-3 targeting peptide, characterized in that: The VEGFR-3 targeting peptide as claimed in claim 1 is used to prepare a visualization imaging agent.

3. The use of a VEGFR-3 targeting peptide according to claim 2, characterized in that: The visualization developer contains a chelate, which is 68 Ga-DOTA-TMVP1446 or 68 Ga-DOTA-TMVP1448; in 68 The molecular formula of Ga-DOTA-TMVP1446 is 68 Ga-DOTA-CRRALGGGTTS R; or 68 The molecular formula of Ga-DOTA-TMVP1448 is 68 Ga-DOTA-SLIGHGRGRR VR.

4. A method for preparing a visualization developer as claimed in claim 2 or 3, characterized in that: The following steps are involved: (1) synthesizing peptides from DOTA and VEGFR-3 targeting peptides and preparing peptide solutions; (2) Rinse with hydrochloric acid solution 68 Ge / 68 Ga generator, collect eluent A, and detect 68 Radioactive dose of Ga; (3) Mixing the sodium acetate solution and the eluent A evenly, and then mixing all of the mixture with the polypeptide solution to obtain a mixed solution, and heating the mixture to obtain a visualization developer.

5. The method for preparing a visualization developer according to claim 4, characterized in that: The concentration of the polypeptide in the polypeptide solution is 0.8-1.2 mg / mL.

6. The method for preparing a visualization developer according to claim 5, characterized in that: The concentration of the hydrochloric acid solution is 0.1-0.3M; the concentration of the sodium acetate solution is 1.20-1.30M.

7. A method for preparing a visualization developer according to claim 6, characterized in that: The volume ratio of the polypeptide solution, the hydrochloric acid solution and the sodium acetate solution is (0.009-0.011):(4-6):(0.090-0.095).

8. A method for preparing a visualization developer according to claim 7, characterized in that: The heating reaction in step (3) is specifically: heating the reaction in a 100° C. metal bath for 15 minutes.

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