CN117486975A - TL-02 polypeptide targeting gastric cancer, polypeptide conjugate and application thereof - Google Patents
TL-02 polypeptide targeting gastric cancer, polypeptide conjugate and application thereof Download PDFInfo
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- CN117486975A CN117486975A CN202311453641.8A CN202311453641A CN117486975A CN 117486975 A CN117486975 A CN 117486975A CN 202311453641 A CN202311453641 A CN 202311453641A CN 117486975 A CN117486975 A CN 117486975A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Genetics & Genomics (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to the technical field of fluorescent contrast agents, and particularly discloses a TL-02 polypeptide targeting gastric cancer, a polypeptide conjugate and application thereof, wherein the polypeptide is the TL-02 polypeptide, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1. The fluorescent contrast agent is prepared by connecting-COOH in near infrared one-region fluorescent dye MPA and amino of tryptophan Trp in TL-02 polypeptide through an amide bond, can target various stomach cancers in vivo, and has higher tumor/normal tissue contrast.
Description
Technical Field
The invention relates to the technical field of fluorescent contrast agents, in particular to a TL-02 polypeptide targeting gastric cancer, a polypeptide conjugate thereof and application thereof.
Background
Gastric Cancer (Gastric Cancer), originating from Gastric mucosal epithelium, is one of the most common digestive tract malignant tumors worldwide, and is prone to lymphatic, peritoneal and liver metastasis. Despite the diversity of gastric cancer treatment approaches, surgical resection remains the core means of treating gastric cancer, even the only means of radically treating gastric cancer. Omission of microscopic lesions and incomplete excision of cancerous tissue results in higher missed diagnosis rates and lower negative cut edge rates. In gastric cancer surgery, how to precisely define the boundary between a tumor and normal tissue in real time is a great challenge for surgery. Aiming at the clinical challenge, the fluorescence imaging navigation technology provides a feasible countermeasure for visualizing the tumor and the surrounding healthy tissues in real time and accurately in operation.
Near Infrared (NIR) fluorescence surgical navigation is an optical imaging technology without ionizing radiation and with high sensitivity, is one of the most promising imaging technologies developed recently, and provides opportunities for clearer and accurate distinction of lesion tissues from normal tissues, reduction of incisional edge positive rate and minimization of anesthesia time. The NIR fluorescence can capture specific molecules at tissue level, cell level and even subcellular level, can be used for drawing the outline of the tumor in real time in operation, and provides an objective and effective tool for the operation navigation and treatment research of malignant tumor. The technique has higher sensitivity than visual and palpation feedback based on subjective, and can scan a larger tissue surface, which is beneficial to finding more hidden lesions. The NIR fluorescence has the advantages of small self-fluorescence, less scattering, high signal-to-noise ratio, no visual field interference, deeper tissue penetration and the like, can provide objective and accurate tumor boundary real-time positioning for surgical operation, and is important for improving the negative edge cutting rate and survival rate of gastric cancer patients.
Since the first report of Kitano et al, japanese scientist in 1994, the laparoscopic distal gastric cancer radical treatment has been widely used clinically through the development of more than 20 years. Laparoscopic gastric cancer resection combined with d1+ or D2 lymph node resection has become a standard surgical procedure for gastric cancer. Indocyanine green (Indocyanine Green, ICG) is the only FDA approved NIR fluorescent dye for clinical use, and its main principles of non-specific passive targeting are perfusion differences, high permeability and long retention Effects (EPR). ICG is used as a new fluorescent operation navigation contrast agent to obtain an pleasing effect in tumor incising edges such as liver cancer, breast cancer, non-small cell lung cancer and the like and cleaning sentinel lymph nodes. With the successful application of ICG fluorescence imaging technology on laparoscopic equipment, ICG guided negative incisal edge and lymph node cleaning of laparoscopic gastric cancer becomes a new exploration direction and shows wide clinical application prospect.
Besides ICG, there are few reports of other NIR fluorescent probes for gastric carcinoma cutting and lymph node cleaning. Although few studies report NIR probes constructed based on quantum dots and gastric cancer specific proteins, the NIR probes are mainly concentrated on detection of in vitro animal models and human in vitro specimens, and biosafety is the biggest bottleneck restricting clinical transformation. There are also scholars to identify gastric cancer during operation by using folic acid targeted OTL38 NIR probe, but the sensitivity and specificity of the probe OTL38 to gastric cancer need further experimental study. Although ICG is the standard NIR dye recommended for gastric cancer laparoscopy, ICG has significant limitations in guiding gastric cancer negative cutting edges and lymph node cleaning. (1) The passive targeting of ICG resulted in high false negative rates (35.3% -60.0%) in gastric cancer negative cut-off; (2) It is possible that ICG fails to enter the lymph nodes, resulting in an accuracy of lymph node visualization of about 70.0% to 95.2%; (3) ICG accumulates not only in cancerous tissue, but also in inflammatory tissue and wound areas; (4) ICG needs to be injected into cancer Zhou Nianmo or gastric serosa under the guidance of a gastroscope, and the risk of ICG overflow caused by gastric wall penetration exists, so that the observation and judgment in operation are affected;
therefore, the development of a gastric cancer specific targeted NIR fluorescent probe to break through ICG nonspecific uptake restriction for guiding gastric cancer accurate negative incisal margin and lymph node cleaning in operation is urgent.
Disclosure of Invention
The TL-02 polypeptide provided by the invention can target and identify various stomach cancers in vivo, shows high tumor/normal tissue signal-to-noise ratio, and can be used for preparing gastric cancer fluorescent contrast agents.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a TL-02 polypeptide targeting gastric cancer, and the amino acid sequence of the TL-02 polypeptide is shown as SEQ ID NO. 1.
The invention also provides a biological material containing a nucleic acid molecule encoding the TL-02 polypeptide, which is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineering bacterium, or a transgenic cell line.
The invention also provides a polypeptide conjugate, which is obtained by coupling the TL-02 polypeptide with a carrier;
the carrier is at least one of cytokines, radioactive elements, carrier proteins, antibodies, enzymes, fluorophores, quantum dots or chromophores with high absorptivity.
Preferably, the fluorescent group is a near infrared first-region fluorescent dye and/or a near infrared second-region fluorescent dye.
Preferably, the near infrared one-region fluorescent dye comprises one or more of MPA, IRDye800, IR820, cy7.5, cy7, ICG and Cy5.5, and the near infrared two-region fluorescent dye comprises FD1080 and/or CH1055.
Preferably, the polypeptide conjugate is amide linked to the amino group of tryptophan Trp in the TL-02 polypeptide by-COOH in the near infrared one-region fluorescent dye MPA, the chemical structure of which is shown in fig. 1.
The invention also provides application of the TL-02 polypeptide or the polypeptide conjugate in preparing medicines for treating gastric cancer or developing agents for gastric cancer.
Preferably, the imaging agent is a tumor boundary accurate localization and/or intra-operative navigation fluorescent imaging agent.
The invention has the following beneficial effects:
1. the targeting polypeptide MPA-TL-02 polypeptide provided by the invention can target various stomach cancers in vivo, has higher tumor/normal tissue contrast ratio, and has a prospect for guiding accurate excision of clinical stomach cancer operation; the MPA-TL-02 polypeptide consists of natural amino acids, is rapid in metabolism in vivo, has high safety and has clinical application prospect.
2. The targeting polypeptide MPA-TL-02 provided by the invention can be used for preparing gastric cancer contrast agents and can provide an effective means for gastric cancer diagnosis.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a chemical structure diagram of a fluorescent contrast agent MPA-TL-02;
FIG. 2 is a mass spectrum of polypeptide TL-02;
FIG. 3 is a mass spectrum of fluorescent contrast agent MPA-TL-02;
FIG. 4 is a 6h fluorescence imaging diagram of the prepared fluorescent contrast agent MPA-TL-02 in a tumor-bearing mouse body;
in the figure, A is fluorescence imaging of gastric cancer MGC-803; b is gastric cancer SGC-7901 fluorescence imaging; c is fluorescence imaging of gastric cancer MKN45.
Detailed Description
The invention provides a TL-02 polypeptide targeting gastric cancer, and the amino acid sequence of the TL-02 polypeptide is shown as SEQ ID NO. 1.
The invention also provides a biological material containing the TL-02 polypeptide nucleic acid molecule, wherein the biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, engineering bacteria or a transgenic cell line.
The invention also provides a polypeptide conjugate, which is obtained by coupling the TL-02 polypeptide with a carrier; the carrier is at least one of cytokines, radioactive elements, carrier proteins, antibodies, enzymes, fluorophores, quantum dots or chromophores with high absorptivity.
In the present invention, the carrier is a fluorescent group.
In the invention, the fluorescent group is a near infrared first-region fluorescent dye and/or a near infrared second-region fluorescent dye; preferably, the near infrared one-region fluorescent dye comprises one or more of MPA, IRDye800, IR820, cy7.5, cy7, ICG and Cy5.5, and the near infrared two-region fluorescent dye comprises one or two of FD1080 and CH1055, more preferably MPA.
In the present invention, the polypeptide conjugate is connected with the amino group of tryptophan Trp in the TL-02 polypeptide through-COOH in near infrared one-region fluorescent dye MPA by an amide bond, and the chemical structure is shown in figure 1.
The invention also provides application of the TL-02 polypeptide or the polypeptide conjugate in preparing medicines for treating gastric cancer or developing agents for gastric cancer.
In the present invention, the imaging agent is a tumor boundary accurate localization and/or intra-operative navigation fluorescent imaging agent.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1A gastric cancer-targeting TL-02 polypeptide
The TL-02 polypeptide is linear decapeptide, the TL-02 polypeptide sequence is shown in SEQ ID NO.1, and the polypeptide is synthesized by a solid phase method.
SEQ ID NO.1：
Trp-Tyr-Val-His-Thr-Gln-Ser-Ile-Met-Arg(WYVHTQSIMR)。
Example 2A fluorescent contrast agent MPA-TL-02
The synthesis process of the fluorescent contrast agent MPA-TL-02 comprises the following steps:
1. 750mg of Fmc-Arg (Pbf) -Wang Resin with Loading of 0.40mmol/g is selected and put into a reaction column, and Fmoc protecting groups are removed after swelling. Fmoc-Met-OH 303.6mg was weighed, pyBop (benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate) 468.2mg,DIPEA 3.5mL was dissolved in 15mL of DMF at 0℃and added to a reaction column to react at room temperature for 1 hour after complete dissolution, and the reaction progress was judged by detecting negative with ninhydrin assay.
After the coupling reaction, the reaction solution was drained, washed with 30mL of DMF, 10mL of 20% hexahydropyridine-DMF solution was reacted for 5min, once with DMF, 10mL of 20% hexahydropyridine-DMF solution was added again after the washing was completed, drained after 10min of reaction, DMF was washed 3 times, DCM (dichloromethane) was washed 2 times, DMF was washed 1 time. Coupling is carried out from the C end to the N end in sequence according to the polypeptide sequence until Fmoc-Trp (OtBu) -OH is reached, and all amino acids are protected by Fmoc to alpha amino; after the coupling reaction was completed, the reaction mixture was drained, washed 3 times with 15mL of DMF, dried after 3 times of methanol washing, and the resin was weighed 1323.2mg.
2. About 329.2mg of polypeptide with all side chain protecting groups removed was obtained by reaction of the cleavage solution (TFA: triisopropylsilane: water=95:2.5:2.5) with a linear peptide resin; the crude aqueous solution of linear peptide was filtered through a 0.45 μm filter and the crude peptide was purified using a high performance liquid chromatography apparatus: and (3) through a DAC-HB50 dynamic axial compression column, the mobile phase A is a trifluoroacetic acid aqueous solution with the mass percentage concentration of 0.05%, and the mobile phase B is a trifluoroacetic acid acetonitrile solution with the mass percentage concentration of 0.05%, gradient elution, separation and purification are carried out, an ultraviolet detector is adopted to detect a sample, and a peptide solution of a target peak is collected in a segmented manner. And (3) performing high-efficiency liquid phase purification to obtain 50mL of finished peptide liquid with the purity of more than 98%, and performing rotary evaporation concentration to obtain 35mL of liquid. The liquid was pre-lyophilized, lyophilized and weighed to give 56mg of product. ESI-MS identification of the product as target polypeptide WYVHTQSIMR, [ M+H ]] + = 1321.23 and [ m+h] 2+ = 661.32, as shown in fig. 2.
3. 5.0mg of TL-02 polypeptide pure was dissolved in 500. Mu.L of DMF and then added with 1.5 times molar amount of near infrared dye MPA-COOH,1.5 times molar amount of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) and 5 times molar amount of N, N-Diisopropylethylamine (DIPEA) and reacted at room temperature in the dark for 1 hour. After the reaction is finished, MPA-TL-02 reaction liquid is purified by preparative chromatography, liquid with qualified purity is separated, and the liquid is collected, evaporated and freeze-dried in a rotary way, and is confirmed by ESI-MS to be a target product (MPA-TL-02), [ M+H ]] 3+ = 745.12 and [ m+h] 4+ = 559.16, as shown in fig. 3.
Example 3 fluorescent imaging of fluorescent contrast agent MPA-TL-02 in gastric cancer MGC-803 tumor bearing mice.
The fluorescent contrast agent MPA-TL-02 prepared in example 2 was prepared and prepared as a physiological saline solution (100 nmol/mL), 0.1mL (about 10 nmol) was injected into tail veins of 3 gastric cancer MGC-803 tumor-bearing mice (weighing about 25 g) respectively, and optical signal acquisition was performed at 1h, 2h, 4h, 6h, 8h, 10h and 12h after administration. The distribution of fluorescent compounds in tumor-bearing mice and the targeted enrichment in tumors were observed. The results after 6h of administration are shown as A in FIG. 4, which shows that the fluorescent contrast agent MPA-TL-02 can target gastric cancer MGC-803.
Example 4 fluorescent contrast agent MPA-TL-02 fluorescence imaging in gastric cancer SGC-7901 tumor bearing mice.
The fluorescent contrast agent MPA-TL-02 prepared in example 2 was prepared as a physiological saline solution (100 nmol/mL), 0.1mL (about 10 nmol) was injected into tail veins of 3 gastric cancer SGC-7901 tumor-bearing mice (weighing about 25 g) respectively, and optical signal acquisition was performed at 1h, 2h, 4h, 6h, 8h, 10h and 12h after administration. The distribution of fluorescent compounds in tumor-bearing mice and the targeted enrichment in tumors were observed. The results after 6h of administration are shown as B in FIG. 4, and the results show that the fluorescent contrast agent MPA-TL-02 can target gastric cancer SGC-7901.
Example 5 fluorescent contrast agent MPA-TL-02 fluorescence imaging in gastric cancer MKN45 tumor-bearing mice.
The fluorescent contrast agent MPA-TL-02 prepared in example 2 was prepared as a physiological saline solution (100 nmol/mL), 0.1mL (about 10 nmol) was injected into tail veins of 3 gastric cancer MKN45 tumor-bearing mice (about 22 g in weight) respectively, and optical signal acquisition was performed at 1h, 2h, 4h, 6h, 8h, 10h and 12h after administration. The distribution of fluorescent compounds in tumor-bearing mice and the targeted enrichment in tumors were observed. The results after 6h of administration are shown as C in FIG. 4, and the results indicate that the fluorescent contrast agent MPA-TL-02 can target gastric cancer MKN45.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. The TL-02 polypeptide targeting gastric cancer is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.
2. A biological material comprising a nucleic acid molecule encoding the TL-02 polypeptide of claim 1, wherein said biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineering bacterium, or a transgenic cell line.
3. A polypeptide conjugate, wherein the polypeptide conjugate is obtained by coupling the TL-02 polypeptide of claim 1 to a carrier; the carrier is at least one of cytokines, radioactive elements, carrier proteins, antibodies, enzymes, fluorophores, quantum dots or chromophores with high absorptivity.
4. The polypeptide conjugate of claim 3 wherein the fluorescent moiety is a near infrared one-region fluorescent dye and/or a near infrared two-region fluorescent dye.
5. The polypeptide conjugate of claim 4 wherein the near infrared one-region fluorescent dye comprises one or more of MPA, IRDye800, IR820, cy7.5, cy7, ICG, and Cy5.5 and the near infrared two-region fluorescent dye comprises FD1080 and/or CH1055.
6. The polypeptide conjugate of claim 5, wherein the polypeptide conjugate is amide linked to the amino group of tryptophan Trp in the TL-02 polypeptide by-COOH in the near infrared one-region fluorescent dye MPA, and has the chemical structure shown below:
7. use of a TL-02 polypeptide of claim 1 or a polypeptide conjugate of any one of claims 3-6 in the manufacture of a medicament for treating gastric cancer or an imaging agent for gastric cancer.
8. The use of claim 7, wherein the imaging agent is a tumor boundary pinpoint and/or an intra-operative navigator fluorescence imaging agent.
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