A tumor-targeting polypeptide V-1-GGGK, its polypeptide conjugate and its application

Abstract

The present invention relates to the technical field of fluorescent contrast agents, and specifically discloses a tumor-targeting polypeptide V-1-GGGK, a polypeptide conjugate thereof and an application thereof. The polypeptide is a V-1-GGGK polypeptide, and its amino acid sequence is shown in SEQ ID NO.1. The molecular probe is a near-infrared fluorescent dye covalently coupled to the polypeptide V-1-GGGK, and can be used as a tumor-targeting fluorescent molecular probe for real-time visualization of tumors and tumor boundaries, and has the advantage of assisting doctors to improve surgical accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent contrast agents, and in particular to a tumor targeting polypeptide V-1-GGGK, a polypeptide conjugate thereof and applications thereof. Background Art

[0002] Although conventional medical imaging methods can be used before and during surgery, the real-time distinction between tumors and their margins and surrounding normal tissues during surgery remains a major challenge for surgeons. To address this clinical challenge, fluorescence imaging navigation technology combined with tumor-targeted molecular probes provides a practical means to accurately visualize tumors and their surrounding healthy tissues in real time during surgery. Near-infrared fluorescence surgical navigation is a non-ionizing radiation, highly sensitive optical imaging technology. It is one of the most promising imaging technologies developed recently, providing an opportunity to more clearly and accurately distinguish diseased tissues from normal tissues, reduce the positive rate of resection margins, and minimize anesthesia time. Near-infrared fluorescence can capture specific molecules at the tissue level, cellular level, and even subcellular level, and can depict the contours of tumors in real time during surgery, providing an objective and effective tool for surgical navigation and treatment research of malignant tumors. This technology has higher sensitivity than subjective visual inspection and palpation feedback, and can scan a larger tissue surface, which is conducive to the discovery of more hidden lesions. Near-infrared fluorescence, with its advantages of low autofluorescence, less scattering, high signal-to-noise ratio, no field of view interference and deeper tissue penetration, can provide objective and accurate real-time positioning of tumor boundaries for surgical operations, which is crucial to improving the negative resection margin rate and survival rate of cancer patients.

[0003] Notable candidates in clinical trials include the fluorescently conjugated anti-vascular endothelial growth factor (VEGFR) antibody Bevacizumab-IRDye800CW and the anti-carcinoembryonic antigen (CEA) antibody SGM-101, which show great promise for tumor fluorescence surgical navigation. However, antibody-conjugated probes have inherent limitations, such as poor tissue penetration and slow pharmacokinetics due to their large size, resulting in prolonged imaging time after administration. In addition, problems such as enhanced permeability and retention effects, neonatal Fc receptor-mediated recycling, and nonspecific accumulation of antibody-fluorophore conjugates in normal tissues can lead to false-positive results. Peptides combine the advantages of small molecule compounds and antibodies, including strong tissue penetration, high affinity, rapid metabolism, low immunogenicity, and low nonspecific binding. Peptides provide a valuable alternative to antibodies due to their high selectivity, effectiveness, relative safety, and good tolerability. Peptides have also been widely recognized for their rapid development in cancer diagnosis and treatment.

[0004] The c-Met receptor is regulated by hepatocyte growth factor (HGF) and plays crucial roles in a variety of cell types, such as inducing growth, survival, and migratory behaviors. The HGF / c-Met signaling pathway is crucial for controlling a variety of biological processes, and its dysregulation contributes to cancer. Although many c-Met-targeted probes for tumor imaging have been developed and validated in preclinical or clinical settings, most have been designed to monitor c-Met expression rather than activation. Considering that c-Met can be activated in cancer by both HGF-dependent and HGF-independent mechanisms, the assessment of HGF-dependent c-Met activation using molecular probes targeting active HGF is essential for patient selection for HGF / c-Met inhibitors, detection of early metastatic cancer, and optical surgical navigation of HGF-positive tumors. Overexpression of c-Met and HGF is associated with poor prognosis and can promote tumor angiogenesis, growth, invasion, and metastasis, making c-Met and HGF valuable targets for tumor diagnosis and therapy.

[0005] Therefore, it is of great significance to develop tumor-targeted near-infrared fluorescent probes based on HGF protein for tumor imaging and guiding precise negative tumor resection margins and lymph node dissection during surgery. Summary of the invention

[0006] The present invention provides a tumor targeting polypeptide V-1-GGGK, a polypeptide conjugate thereof and applications thereof. The V-1-GGGK polypeptide provided by the present invention can target and recognize a variety of tumors in vivo and exhibit a high tumor / normal tissue signal-to-noise ratio, and can be used to prepare a tumor fluorescent contrast agent.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a tumor targeting polypeptide V-1-GGGK, whose amino acid sequence is shown in SEQ ID NO.1.

[0009] The present invention also provides a polypeptide conjugate, which is obtained by coupling the targeting polypeptide V-1-GGGK with a fluorescent dye; the fluorescent dye is selected from at least one of a fluorescent group, a quantum dot or a high absorption coefficient chromophore.

[0010] Preferably, the fluorescent group is a near-infrared region 1 fluorescent dye and / or a near-infrared region 2 fluorescent dye.

[0011] Preferably, the near-infrared region 1 fluorescent dye includes one or more of MPA, IRDye800, IR820, Cy7.5, Cy7, ICG and Cy5.5, and the near-infrared region 2 fluorescent dye includes FD1080 and / or CH1055.

[0012] Preferably, the polypeptide conjugate is connected to the lysine side chain amino group in the V-1-GGGK polypeptide through an amide bond via the -COOH in the near-infrared region 1 fluorescent dye MPA, and its chemical structure is shown below:

[0013]

[0014] The present invention also provides a use of the V-1-GGGK polypeptide or the polypeptide conjugate in the preparation of anti-tumor drugs, tumor-targeting drugs or tumor imaging agents.

[0015] Preferably, the imaging agent is a fluorescent imaging agent for precise positioning of tumors or tumor boundaries and / or intraoperative navigation.

[0016] Preferably, the tumor includes lung cancer, colorectal cancer or gastric cancer.

[0017] The present invention has the following beneficial effects:

[0018] 1. The targeted V-1-GGGK polypeptide provided by the present invention can target multiple tumors in vivo and has a high tumor / normal tissue contrast, and has the prospect of being used to guide precise resection of clinical tumor surgery; the V-1-GGGK polypeptide is composed of natural amino acids, is rapidly metabolized in vivo, has high safety, and has clinical application prospects.

[0019] 2. The targeting polypeptide V-1-GGGK provided by the present invention can be used to prepare tumor contrast agents, and can provide an effective means for tumor diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The chemical structure diagram of the fluorescent contrast agent V-1-GGGK-MPA;

[0022] Figure 2 is the mass spectrum of polypeptide V-1-GGGK in Example 2;

[0023] Figure 3 is the mass spectrum of the fluorescent contrast agent V-1-GGGK-MPA in Example 2;

[0024] Figure 4 The fluorescence imaging images of the fluorescent contrast agent V-1-GGGK-MPA in Examples 3 to 6 in various colorectal cancer-bearing mice;

[0025] Figure 5 The fluorescence imaging images of the fluorescent contrast agent V-1-GGGK-MPA in Examples 3 to 6 in vitro in various colorectal cancer-bearing mice;

[0026] Figure 6 is the signal-to-noise ratio of tumor and normal tissue of the fluorescent contrast agent V-1-GGGK-MPA in various colorectal cancer-bearing mice in Examples 3 to 6;

[0027] Figure 7 These are the fluorescence imaging images of the fluorescent contrast agent V-1-GGGK-MPA in Examples 7 to 9 in lung cancer and gastric cancer-bearing mice for 7 hours. DETAILED DESCRIPTION

[0028] The present invention provides a tumor targeting polypeptide V-1-GGGK, whose amino acid sequence is shown in SEQ ID NO.1.

[0029] The present invention also provides a polypeptide conjugate, which is obtained by coupling the targeting polypeptide V-1-GGGK with a fluorescent dye; the fluorescent dye is selected from at least one of a fluorescent group, a quantum dot or a high absorption coefficient chromophore.

[0030] In the present invention, the fluorescent group is a near-infrared region 1 fluorescent dye and / or a near-infrared region 2 fluorescent dye. The near-infrared region 1 fluorescent dye is preferably one or more of MPA, IRDye800, IR820, Cy7.5, Cy7, ICG and Cy5.5. The near-infrared region 2 fluorescent dye is preferably FD1080 and / or CH1055, and more preferably MPA.

[0031] In the present invention, the polypeptide conjugate is connected to the lysine side chain amino group in the V-1-GGGK polypeptide through an amide bond via the -COOH in the near-infrared region 1 fluorescent dye MPA. Its chemical structure is as follows: Figure 1 shown.

[0032] The present invention also provides a use of the V-1-GGGK polypeptide or the polypeptide conjugate in the preparation of anti-tumor drugs, tumor-targeting drugs or tumor imaging agents.

[0033] In the present invention, the imaging agent is a fluorescent imaging agent for precise positioning of tumors or tumor boundaries and / or intraoperative navigation.

[0034] In the present invention, the tumor includes lung cancer, colorectal cancer or gastric cancer.

[0035] 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.

[0036] Example 1

[0037] A tumor-targeting V-1-GGGK peptide

[0038] The V-1-GGGK polypeptide is a linear nonapeptide, and the V-1-GGGK polypeptide sequence is shown in SEQ ID NO. 1. This polypeptide was commissioned to Hangzhou Gutuo Biotechnology Co., Ltd. to synthesize it through a standard solid phase method.

[0039] SEQ ID NO.1:

[0040] Ac-Nle-Tyr(3-I)-Leu-Hyp-Oic-Gly-Gly-Gly-Lys-NH2

[0041] Example 2

[0042] Synthesis of a Fluorescent Contrast Agent V-1-GGGK-MPA

[0043] 1. Preparation of polypeptide V-1-GGGK

[0044] 1.1 Preparation of Fmoc-Lys(Boc)-amino resin

[0045] 1) Select 66.7 mg of Ramage Amide AM resin with a loading of 0.30 mmoL / g and place it into the reaction column to remove the Fmoc protecting group.

[0046] 2) Weigh 281.12 mg of Fmoc-Lys(Boc)-OH, 312.12 mg of PyBop (benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate), and 0.21 mL of DIEA (N,N-diisopropylethylamine) and dissolve them in 15 mL of DMF (N,N-dimethylformamide) at 0°C. After complete dissolution, add them to the reaction column and react at room temperature for 1 hour. The reaction progress is judged by the negative result of the ninhydrin detection method. After the coupling reaction is completed, the reaction solution is drained, washed with 15 mL of DMF, 10 mL of 20% hexahydropyridine / DMF solution, reacted for 5 minutes, washed once with DMF, and after washing, 10 mL of 20% hexahydropyridine / DMF solution is added again. After reacting for 10 minutes, drain, wash 3 times with DMF, wash 2 times with DCM, and wash once with DMF to obtain Fmoc-Lys(Boc)-amino resin.

[0047] Preparation of 1.2V-1-GGGK linear peptide resin

[0048] Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Oic-OH, Fmoc-Hyp(tBu)-OH and Fmoc-Tyr(3-I)-OH were added to the above-mentioned Fmoc-Lys(Boc)-amino resin in sequence from C-terminus to N-terminus according to the polypeptide sequence for coupling until Fmoc-Nle-OH was obtained, and the Fmoc protecting group was removed using a piperidine / DMF mixture.

[0049] The side chain protecting groups of His, Hyp and Lys are Trt (trityl), tBu (tert-butyl) and Boc (tert-butyloxycarbonyl), respectively; all amino acids are protected with Fmoc (9-fluorenylmethoxycarbonyl) at the α-position amino group; after the coupling reaction, 1 mL of acetic anhydride is used to block the N-terminus of the peptide chain, the reaction solution is drained, washed 3 times with 15 mL of DMF, washed 3 times with methanol and then dried, and 410.25 mg of V-1-GGGK linear peptide resin is weighed.

[0050] Preparation of crude 1.3V-1-GGGK peptide

[0051] 2 mL of lysis solution (TFA: triisopropylsilane: water = 95:2.5:2.5) was reacted with 410 mg of linear peptide resin for 2 hours, and the crude peptide was precipitated with 40 mL of icy ether. The crude peptide was then washed with 20 mL of ether three times, and the solid crude peptide was vacuum dried to obtain about 252.12 mg of V-1-GGGK linear crude peptide with all side chain protecting groups removed.

[0052] Preparation of 1.4V-1-GGGK boutique peptide

[0053] 1) Filter the crude linear peptide aqueous solution using a 0.45 μm filter membrane.

[0054] 2) Purify the filtered crude linear peptide aqueous solution using a high performance liquid chromatography instrument: Use a DAC-HB50 dynamic axial compression column, the mobile phase A is a 0.05% trifluoroacetic acid aqueous solution, and the mobile phase B is a 0.05% trifluoroacetic acid acetonitrile solution. Gradient elution separation and purification is performed, and the sample is detected using a UV detector, and the peptide solution of the target peak is collected in segments.

[0055] 3) After high performance liquid phase purification, 30 mL of finished peptide liquid with a purity greater than 85% to 90% was obtained, and 15 mL of liquid was obtained after rotary evaporation concentration.

[0056] 4) The liquid was pre-lyophilized and freeze-dried to finally obtain 19.7 mg of the fine peptide. The product was identified by ESI-MS as the target peptide Ac-Nle-Y(3-I)LH-Hyp-Oic-GGGK-NH2, [M+H] +=1276.49 and [M+H] 2+ =638.94, such as Figure 2 shown.

[0057] 2. Synthesis of fluorescent contrast agent V-1-GGGK-MPA

[0058] 10.0 mg of V-1-GGGK fine peptide was dissolved in 200 μL DMF, and then 1.5 times the molar amount of near-infrared dye MPA-COOH, 1.5 times the molar amount of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) and 5 times the molar amount of N, N-diisopropylethylamine (DIPEA) were added, and the reaction was carried out at room temperature in the dark for 1 hour. After the reaction was completed, the V-1-GGGK-MPA reaction solution was purified by preparative chromatography to separate a liquid with a purity of 96.5. After collecting and lyophilizing, 8.65 mg of the product was obtained, which was confirmed by ESI-MS to be the target product V-1-GGGK-MPA, [MH] 2- =1091.83 and [MH] 3- =727.55, such as Figure 3 shown.

[0059] Example 3

[0060] Fluorescence imaging of the fluorescent contrast agent V-1-GGGK-MPA in colorectal cancer SW480 tumor-bearing mice in vivo and in vitro.

[0061] Take the fluorescent contrast agent V-1-GGGK-MPA prepared in Example 2 and prepare it into a physiological saline solution (150nmoL / mL). Take 0.1mL (about 15nmoL) and inject it into the tail vein of 3 colorectal cancer SW480 tumor-bearing mice (weighing about 25 grams), and collect optical signals 1 to 12 hours after administration. Observe the distribution of fluorescent compounds in tumor-bearing mice and the targeted enrichment in tumors, and euthanize mice 12 hours after imaging. Perform in vitro imaging of tumors and major tissues and organs, and calculate the fluorescence signal-to-noise ratio of tumors and major organ tissues. The results of in vivo fluorescence imaging are shown in Figures 4-5 The results showed that the fluorescent contrast agent V-1-GGGK-MPA can target colorectal cancer SW480. The fluorescence quantitative results of tumor / major organ tissues are shown in Figure 6 , indicating that the contrast agent V-1-GGGK-MPA can identify colorectal cancer SW480 tumors with a higher tumor / normal tissue signal-to-noise ratio.

[0062] Example 4

[0063] Fluorescence imaging of the fluorescent contrast agent V-1-GGGK-MPA in vitro and in vivo in colorectal cancer SW480 tumor-bearing mice after blockade.

[0064] Take 0.2 mL (about 1.5 μmoL) of pure peptide V-1-GGGK from Example 2 and inject it into three colorectal cancer SW480 tumor-bearing mice 30 minutes in advance, then take the prepared fluorescent contrast agent V-1-GGGK-MPA and prepare it into physiological saline solution (150nmoL / mL), take 0.1 mL (about 15nmoL) and inject it into the tail vein of three colorectal cancer SW480 tumor-bearing mice (weighing about 25 grams), and collect optical signals 1 to 12 hours after administration. Observe the distribution of fluorescent compounds in tumor-bearing mice and the targeted enrichment in tumors, and euthanize mice 12 hours after imaging, perform in vitro imaging of tumors and major tissues and organs, and calculate the fluorescence signal-to-noise ratio of tumors and major organ tissues. The results of in vivo and in vitro fluorescence imaging are shown in Figures 4-5 It can be seen that after being blocked by a large amount of pure peptide V-1-GGGK, the fluorescence signal of tumor tissue was significantly reduced, indicating that V-1-GGGK-MPA is specifically targeted to colorectal cancer SW480. Further fluorescence quantitative results of tumor / major organ tissues ( Figure 6 ) also demonstrated that the contrast agent V-1-GGGK-MPA is specifically targeted to colorectal cancer SW480 tumors.

[0065] Example 5

[0066] Fluorescence imaging of the fluorescent contrast agent V-1-GGGK-MPA in colorectal cancer HT29 tumor-bearing mice in vivo and in vitro.

[0067] Take the fluorescent contrast agent V-1-GGGK-MPA prepared in Example 2 and prepare it into a physiological saline solution (150nmoL / mL). Take 0.1mL (about 15nmoL) and inject it into the tail vein of 3 colorectal cancer HT29 tumor-bearing mice (weighing about 25 grams), and collect optical signals 1 to 12 hours after administration. Observe the distribution of fluorescent compounds in tumor-bearing mice and the targeted enrichment in tumors, and euthanize mice 12 hours after imaging. Perform in vitro imaging of tumors and major tissues and organs, and calculate the fluorescence signal-to-noise ratio of tumors and major organ tissues. The results of in vivo and in vitro fluorescence imaging are shown in Figures 4-5 The results showed that the fluorescent contrast agent V-1-GGGK-MPA can target colorectal cancer HT29. The fluorescence quantitative results of tumor / major organ tissues are shown in Figure 6 , indicating that the contrast agent V-1-GGGK-MPA can identify colorectal cancer HT29 tumors with a higher tumor / normal tissue signal-to-noise ratio.

[0068] Example 6

[0069] Fluorescence imaging of the fluorescent contrast agent V-1-GGGK-MPA in vivo and in vitro in colorectal cancer HCT116 tumor-bearing mice.

[0070] Take the fluorescent contrast agent V-1-GGGK-MPA prepared in Example 2 and prepare it into a physiological saline solution (150nmoL / mL), take 0.1mL (about 15nmoL) and inject it into the tail vein of 3 colorectal cancer HCT116 tumor-bearing mice (weighing about 25 grams), and collect optical signals 1 to 12 hours after administration. Observe the distribution of fluorescent compounds in tumor-bearing mice and the targeted enrichment in tumors, and euthanize mice 12 hours after imaging, perform in vitro imaging of tumors and major tissues and organs, and calculate the fluorescence signal-to-noise ratio of tumors and major organ tissues. The results of in vivo and in vitro fluorescence imaging are shown in Figures 4-5 The results showed that the fluorescent contrast agent V-1-GGGK-MPA can target colorectal cancer HCT116. The fluorescence quantitative results of tumor / major organ tissues are shown in Figure 6 , indicating that the contrast agent V-1-GGGK-MPA can identify colorectal cancer HCT116 tumors with a high tumor / normal tissue signal-to-noise ratio.

[0071] Example 7

[0072] Fluorescence imaging of the fluorescent contrast agent V-1-GGGK-MPA in mice bearing lung cancer A549.

[0073] The fluorescent contrast agent V-1-GGGK-MPA prepared in Example 2 was prepared into a physiological saline solution (150 nmoL / mL). 0.1 mL (about 15 nmoL) was injected into the tail veins of three A549 lung cancer tumor-bearing mice (weighing about 25 grams), and optical signal collection was performed 1 to 12 hours after administration. The distribution of the fluorescent compound in the tumor-bearing mice and the targeted enrichment in the tumor were observed. The in vivo fluorescence imaging results are shown in Figure 7 The results showed that the fluorescent contrast agent V-1-GGGK-MPA can target lung cancer A549.

[0074] Example 8

[0075] Fluorescence imaging of the fluorescent contrast agent V-1-GGGK-MPA in gastric cancer MGC-803 tumor-bearing mice.

[0076] The fluorescent contrast agent V-1-GGGK-MPA prepared in Example 2 was prepared into a physiological saline solution (150 nmoL / mL). 0.1 mL (about 15 nmoL) was injected into the tail vein of three gastric cancer MGC-803 tumor-bearing mice (weight about 25 grams), and optical signal collection was performed 1 to 12 hours after administration. The distribution of the fluorescent compound in the tumor-bearing mice and the targeted enrichment in the tumor were observed. The in vivo fluorescence imaging results are shown in Figure 7 ,The results showed that the fluorescent contrast agent V-1-GGGK-MPA can target gastric cancer MGC-803.

[0077] Example 9

[0078] Fluorescence imaging of the fluorescent contrast agent V-1-GGGK-MPA in gastric cancer SGC-7901 tumor-bearing mice.

[0079] The fluorescent contrast agent V-1-GGGK-MPA prepared in Example 2 was prepared into a physiological saline solution (150 nmoL / mL). 0.1 mL (about 15 nmoL) was injected into the tail vein of three gastric cancer SGC-7901 tumor-bearing mice (weighing about 25 grams), and optical signal collection was performed 1 to 12 hours after administration. The distribution of the fluorescent compound in the tumor-bearing mice and the targeted enrichment in the tumor were observed. The in vivo fluorescence imaging results are shown in Figure 7 , the results showed that the fluorescent contrast agent V-1-GGGK-MPA can target gastric cancer SGC-7901.

[0080] 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 conjugate V-1-GGGK-MPA, characterized in that: The polypeptide conjugate is connected to the lysine side chain amino group in the V-1-GGGK polypeptide through an amide bond via the -COOH in the near-infrared region 1 fluorescent dye MPA. Its chemical structure is shown below:

2. Use of the polypeptide conjugate V-1-GGGK-MPA according to claim 1 in the preparation of a colorectal cancer imaging agent.

3. The use according to claim 2, characterized in that: The imaging agent is a fluorescent imaging agent for precise positioning of tumors or tumor boundaries and / or intraoperative navigation.

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