Cancer targeting peptide and conjugate and application thereof
By developing conjugates of targeted peptides SLV1 and SLV2 with near-infrared fluorescent dyes, the problem of difficult tumor boundary localization in cancer surgery in existing technologies has been solved, enabling precise surgical resection and high-contrast imaging for various cancers and improving surgical outcomes.
Patent Information
- Application Number
- CN202511303433.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-12
- Publication Date
- 2025-12-19
AI Technical Summary
Existing fluorescence imaging technology has difficulty accurately locating the boundary between tumor and normal tissue during surgery for cancers such as pancreatic cancer, liver cancer, breast cancer, colorectal cancer, lung cancer, esophageal cancer, and glioma, resulting in a high rate of missed diagnoses and a low rate of negative resection margins. Furthermore, existing fluorescent probes have limited targeting specificity and cannot be widely applied to various cancer types.
Targeting peptides SLV1 and SLV2 and their conjugates were developed. By binding with near-infrared fluorescent dyes, MPA-PEG2-SLV1 and MPA-PEG2-SLV2 probes were formed, which can specifically target multiple cancer types and be used to accurately locate tumor boundaries and guide surgical resection.
It enables high-contrast tumor/normal tissue identification for various cancer types, improving surgical precision and safety, reducing the rate of missed diagnoses and negative surgical margins, and providing broader prospects for clinical applications.
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Figure CN121159633A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biological medicine, and specifically relates to a cancer targeting peptide and a conjugate thereof and application. BACKGROUND
[0002] Pancreatic cancer, liver cancer, breast cancer, colorectal cancer, lung cancer, esophageal cancer and brain glioma, according to the latest GLOBOCAN 2023 database, the above-mentioned cancers are the main malignant tumors that endanger human beings at present, among which lung cancer, colorectal cancer, esophageal cancer, liver cancer and breast cancer are in the top five in terms of incidence and mortality in China, and breast cancer ranks first among female cancers; and pancreatic cancer has limited early diagnosis technology, and is often found in the late stage, known as the king of cancer; brain glioma is highly malignant and seriously harmful. These cancers seriously endanger the health and life safety of patients and cause huge social and economic burden and loss. Although there are various treatment methods for pancreatic cancer, liver cancer, breast cancer, colorectal cancer, lung cancer, esophageal cancer and brain glioma, surgical resection is still the core means for treating pancreatic cancer, liver cancer, breast cancer, colorectal cancer, lung cancer, esophageal cancer and brain glioma, and even the only means for radical treatment. The missed detection of micro lesions and the incomplete resection of cancer tissues lead to a high missed diagnosis rate and a low negative margin rate. In the surgery of pancreatic cancer, liver cancer, breast cancer, colorectal cancer, lung cancer, esophageal cancer and brain glioma, how to accurately locate detection and accurately define the boundary between tumor and normal tissue in real time is a big challenge in clinical diagnosis and surgical operation. In view of this clinical challenge, fluorescence imaging navigation technology provides a practical means for real-time and accurate visualization of tumors and their surrounding healthy tissues. Near-infrared (NIR) fluorescence surgical navigation is two kinds of non-ionizing radiation, high sensitivity optical imaging technology, which is one of the most promising imaging technologies developed recently, providing opportunities for clearer and more accurate differentiation between diseased and normal tissues, reducing positive margin rates and minimizing anesthesia time. NIR fluorescence can capture specific molecules at the tissue, cellular and even subcellular levels, and can depict tumor contours 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 and palpation feedback and can scan larger tissue surfaces, which is beneficial to find more occult lesions. NIR fluorescence has the advantages of small self-fluorescence, less scattering, high signal-to-noise ratio, no visual interference and deep tissue penetration, which can provide objective and accurate real-time positioning of tumor boundaries for surgical operation, and is crucial for improving the negative margin rate and survival rate of patients with pancreatic cancer, liver cancer, breast cancer, colorectal cancer, lung cancer, esophageal cancer and brain glioma and their subtypes.
[0003] The earliest fluorescence imaging experiment is a contrast agent EC-17 composed of folic acid combined with fluorescein isothiocyanate (emission wavelength 520 nm) for cell transmission surgery of ovarian cancer patients, however, the limited fluorescence penetration and the relatively high spontaneous fluorescence signal of collagen-rich tissues limit its effectiveness. Subsequently, a similar fluorescence tracer OTL-38 targeting folic acid receptors was developed, with an emission wavelength of 796 nm, which increased the tumor detection rate in surgeries including ovarian cancer, lung cancer, kidney and endometrial cancer, and meningioma, but the specificity was not strong enough. In addition to folic acid receptors, certain tumor-specific high-expression cell surface or extracellular matrix proteins such as VEGFR, EGFR, PSMA, CEA, GRPR, Integrins, Cathepsins, MMPs, Cell-surface Chlorotoxin binding proteins, etc. as targets, have been developed and used for intraoperative navigation molecular imaging using specific binding antibodies, minibodies, polypeptides and small molecule compounds, etc. So far, only two fluorescence probes targeting folic acid receptors, indocyanine green (ICG) and Pafolacianine, have been approved by the FDA for marketing for surgical navigation, but the tumor targeting specificity is limited. The total number of probes in the clinical research stage is less than 20, more than half of which are antibodies and their simplifications, and there are problems such as long injection and surgery interval time, and the need for fermentation platform for synthesis, while there are fewer polypeptides and small molecules that are easy to synthesize and low in cost, among which polypeptides are even less. With the addition of new NIR II fluorescence groups with good water solubility and longer wavelength, it is of great significance to discover and develop new polypeptides that can specifically target multiple tumors.
[0004] Therefore, it is urgent to develop a polypeptide carrier and its NIR fluorescence probe that can specifically target multiple cancers such as pancreatic cancer, liver cancer, breast cancer, colorectal cancer, lung cancer, esophageal cancer and brain glioma, in order to break through the limitations of non-specific uptake of ICG and other non-specific uptake, and to be applicable to more cancer types, in order to guide intraoperative precise negative margin and lymph node dissection of multiple cancer types. The invention of a newly discovered and in vivo imaging verified polypeptide carrier that targets tumor cells can achieve precise killing of tumor cells after stably carrying appropriate lethal drugs / materials, thereby enriching the means of precise targeted therapy. SUMMARY
[0005] Therefore, the present application aims to provide a cancer targeting peptide and its conjugate and application. The targeting polypeptides SLV1 and SLV2 provided by the present application can be used to prepare contrast agents for multiple cancer types, providing an effective means for the diagnosis of multiple cancer types.
[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:
[0007] In a first aspect, the present application provides a cancer targeting peptide, the amino acid sequence of which is shown in SEQ ID NO. 1 or SEQ ID NO. 2.
[0008] As a preferred, the cancer targeting peptide is a linear peptide.
[0009] In a second aspect, the present application provides a polypeptide derivative, which is obtained by modifying the N-terminal of the polypeptide with PEG2 and the C-terminal with NH2.
[0010] In a third aspect, the present application provides a polypeptide conjugate, which is obtained by conjugating the polypeptide derivative with a carrier.
[0011] As a preferred, the carrier is selected from one of a cytokine, a radioactive element, a carrier protein, an antibody, an enzyme, a fluorescent group, a quantum dot or a high light absorption coefficient chromophore.
[0012] As a preferred, the fluorescent group is a near-infrared region I fluorescent dye and / or a near-infrared region II fluorescent dye.
[0013] As a preferred, the near-infrared region I fluorescent dye includes one of MPA, IRDye800, IR820, Cy7.5, Cy7, ICG, Cy5.5, and the near-infrared region II fluorescent dye includes FD1080 and / or CH1055.
[0014] Further preferably, the near-infrared region I fluorescent dye is MPA, and the -COOH in the MPA is connected to the amino group of the PEG2 at the N-terminal of the polypeptide derivative by an amide bond, and the chemical structure is as shown in Figure 1 .
[0015] In a third aspect, the present application provides the use of the polypeptide conjugate as described above in the preparation of a medicament for treating cancer, and in the preparation of a cancer imaging agent.
[0016] As a preferred, the cancer includes but is not limited to pancreatic cancer, liver cancer, breast cancer, colorectal cancer, lung cancer, esophageal cancer and brain glioma.
[0017] As a preferred, the cancer imaging agent is used for precise positioning of tumor boundaries and / or intraoperative navigation fluorescence.
[0018] At least the following beneficial technical effects are contained:
[0019] The MPA-PEG2-SLV1, MPA-PEG2-SLV2 polypeptide near-infrared fluorescent probes provided by the application can target various cancers such as pancreatic cancer, liver cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer and brain glioma in the body, have a high tumor / normal tissue contrast ratio, and have the prospect of being used for guiding the precise resection of various cancers such as pancreatic cancer, liver cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer and brain glioma in clinical surgery; the SLV1 and SLV2 polypeptides are composed of natural amino acids, are rapidly metabolized in the body, have high safety, and have the prospect of clinical application.
[0020] The SLV1 and SLV2 polypeptides provided by the application can be used for preparing contrast agents for various cancers such as pancreatic cancer, liver cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer and brain glioma, and can provide an effective means for diagnosing various cancers such as pancreatic cancer, liver cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer and brain glioma. BRIEF DESCRIPTION OF DRAWINGS
[0021] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.
[0022] Figure 1 The figure is a chemical structure diagram of the fluorescent contrast agent MPA-PEG2-SLV1, MPA-PEG2-SLV2;
[0023] Figure 2 The figure is a mass spectrum diagram of the polypeptide derivative PEG2-SLV1, PEG2-SLV2;
[0024] Figure 3 The figure is a mass spectrum diagram of the fluorescent contrast agent MPA-PEG2-SLV1, MPA-PEG2-SLV2;
[0025] Figure 4 The figure is a 4-8h fluorescence imaging diagram of the prepared fluorescent contrast agent MPA-PEG2-SLV1, SLV2 in tumor-bearing mice; in the figure, A is the fluorescence imaging of breast cancer 4T1, esophageal cancer TE1, lung cancer H1299, colorectal cancer HT29, colorectal cancer RKO and colorectal cancer HCT116 displayed by the MPA-PEG2-SLV1 probe; B is the fluorescence imaging of breast cancer 4T1, breast cancer MDA-MB-231, colorectal cancer HCT116, colorectal cancer HT29, liver cancer HCCLM3, pancreatic cancer SW1990 and brain glioma U87 displayed by the MPA-PEG2-SLV2 probe.
[0026] Figure 5 The results of imaging and co-localization of the specific cell membrane binding dye DII, the specific cell nucleus binding dye DPAI and the FITC-PEG2-SLV1 probe with different concentrations of the FITC-PEG2-SLV1 probe with the colorectal cancer HCT116 cells at the same time.
[0027] Figure 6 The results of imaging and co-localization of the specific cell membrane binding dye DII, the specific cell nucleus binding dye DPAI and the FITC-PEG2-SLV1 probe with different concentrations of the FITC-PEG2-SLV1 probe with the RKO cells at the same time.
[0028] Figure 7 The results of the binding of the RKO and lung cancer H1299 cell membrane surfaces and the blocking by SLV1.
[0029] Figure 8 The results of the flow cytometry analysis showing the binding of the FITC-PEG2-SLV1 probe with the colorectal cancer HCT116 cells and the influence of the probe concentration and the blocking by SLV1 and the calculation of the Kd.
[0030] Figure 9 The results of the cell co-localization experiment of the FITC-PEG2-SLV2 probe with the RKO, HCT-116, HT-29 and NCM-460 cells;
[0031] Figure 10 The results of the quantitative analysis of the average fluorescence intensity of the FITC-PEG2-SLV2 probe binding to the cell membranes of the RKO, HCT-116, HT-29 and NCM-460 cells;
[0032] Figure 11 The results of the flow cytometry detection of the binding of the FITC-PEG2-SLV2 probe to the cell membrane surfaces of the colorectal cancer cells RKO, HCT-116, HT-29 and the normal colon epithelial cells NCM-460 and the blocking by SLV2.
[0033] Figure 12 The results of the flow cytometry analysis showing the binding of the FITC-PEG2-SLV2 probe with the colorectal cancer RKO, HCT-116, HT-29 and the normal colon epithelial NCM-460 cells and the influence of the probe concentration and the blocking by SLV2 and the calculation of the Kd. DETAILED DESCRIPTION
[0034] Various illustrative embodiments of the present application are now described in detail below. The following description includes specific details for the purpose of providing a thorough understanding of the present application. However, it will be apparent to those skilled in the art that the present application can be practiced without these specific details. In some instances, well-known structures and functions have not been described in detail in order to avoid obscuring the concept of the present application.
[0035] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also encompassed within the application, subject to any specifically excluded limit in the stated
[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials which are used in connection with the practice of the present application. In the case of conflict between any document incorporated by reference and the present document, the present document controls.
[0037] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.
[0038] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.
[0039] As used herein, the terms "room temperature", "ambient temperature" are intended to mean 25 ± 2 °C, unless otherwise specified.
[0040] The raw materials and instruments used in the following examples of the present application are commercially available, unless otherwise specified.
[0041] Example 1
[0042] The cancer targeting peptides synthesized by solid phase method are respectively denoted as SLV1 and SLV2. The polypeptide sequences of the SLV1 and SLV2 are respectively shown in SEQ ID NO. 1 and SEQ ID NO. 2.
[0043] The cancer targeting peptide is modified with PEG2 at the N-terminus and NH2 at the C-terminus to obtain a cancer targeting peptide derivative.
[0044] SLV1 derivative: PEG2-QNIVHSNGNTY-NH2.
[0045] SLV2 derivative: PEG2-NQLVHSQGQTY-NH2.
[0046] The specific synthesis method is as follows:
[0047] 1. Take 700 mg of Rink Amide MBHA resin with a loading of 0.30 mmol / g, and swell in dichloromethane for 15-20 min in a reaction tube. Perform Fmoc deprotection with 20% piperidine DMF solution for 5 min. Then add Fmoc-Tyr(OtBu)-OH (4 times molar excess compared to the resin) in DMF, pre-activate with HBTU (O-benzotriazol-tetramethyluronium hexafluorophosphate) + HOBT (1-hydroxybenzotriazole) + DIPEA (5 times molar excess compared to the resin), and react with the resin amino group at room temperature for 60 min. Then perform Fmoc deprotection with 20% piperidine for 5 min. After each coupling or deprotection, wash with DMF 4 times. Perform detection by ninhydrin detection method to judge the reaction progress. Repeat the cycle to extend the peptide sequence, and sequentially couple from the C-terminal to the N-terminal until Fmoc-NH-PEG2-COOH. After the coupling reaction is completed, dry the reaction solution, wash with DMF 6 times, wash with methanol 3 times, and then dry and weigh the resin to be 1323.2 mg;
[0048] 2. React the cleavage solution (TFA: triisopropylsilane: water = 95:2.5:2.5) with the linear peptide resin for 3 hours, concentrate the filtrate, add ice-ethanol at -20°C, centrifuge to remove the supernatant, and obtain about 329.2 mg of the crude peptide with all side chain protection groups removed; filter the aqueous solution of the linear peptide crude product with a 0.45 μm filter membrane, and purify the improved crude peptide using a high-performance liquid chromatograph: pass through a DAC-HB50 dynamic axial compression column, use mass percentage 0.05% trifluoroacetic acid aqueous solution as mobile phase A, and mass percentage 0.05% trifluoroacetic acid acetonitrile solution as mobile phase B, perform gradient elution separation and purification, use an ultraviolet detector to detect the sample, and collect the peptide solution of the target peak in sections. After high-performance liquid purification, 50 mL of the finished product peptide liquid with a purity greater than 98% is obtained, which is concentrated by rotary evaporation to obtain 35 mL of liquid. Perform pre-freeze drying and freeze drying on the liquid, and weigh to obtain 56 mg of the product.
[0049] The product is identified by ESI-MS as the target polypeptide as shown in Figure 2 ;
[0050] PEG2-SLV1( Figure 2 A) PEG2-QNIVHSNGNTY-NH2, [M+H] + = 1390.7
[0051] and [M+2H] 2+ = 696.1;
[0052] PEG2-SLV2(Figure 2 B): PEG2-NQLVHSQGQTY-NH2, [M+H] + = 1419.6
[0053] and [M+2H] 2+ = 710.3.
[0054] Example 2
[0055] Preparation of two fluorescent contrast agents MPA-PEG2-SLV1, MPA-PEG2-SLV2
[0056] The molar ratio of near-infrared dye MPA-COOH is 1.5 times the molar amount, 1.5 times the molar amount of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) are dissolved in 500 μL DMF, and the reaction is carried out at room temperature for 1 hour. MPA-NHS is obtained. Then 5.0 mg of SLV1, SLV2 polypeptide derivative pure product and 4 times the molar mass of DIPEA are added and reacted at room temperature for 1 hour. After the reaction is completed, the MPA-PEG2-SLV1, MPA-PEG2-SLV2 reaction solution is purified by preparative chromatography, and the liquid with qualified purity is separated and collected. After rotary evaporation and freeze-drying, it is confirmed by ESI-MS to be the target product: as shown in Figure 3
[0057] (MPA-PEG2-SLV1, Figure 3 A): [M-2H] 2- = 1149.41 and [M-3H] 3- = 765.93;
[0058] (MPA-PEG2-SLV2, Figure 3 B): [M-2H] 2- = 1163.6 and [M-3H] 3- = 775.34.
[0059] Example 3
[0060] Fluorescent contrast agents MPA-PEG2-SLV1, MPA-PEG2-SLV2 in pancreatic cancer, liver cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer and brain glioma in various cancer and its subclass tumor-bearing mice in vivo fluorescence imaging.
[0061] The prepared fluorescent contrast agent MPA-PEG2-SLV1 and MPA-PEG2-SLV2 of Example 2 were prepared into a physiological saline solution (100 nmol / mL), 0.1 mL (about 10 nmol) of which was injected into the tail vein of 3 tumor-bearing mice (about 20 g in weight) of pancreatic cancer, liver cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer and brain glioma and their subtypes, and optical signal collection was performed at 1-8 H after administration. The distribution of the fluorescent compound in the tumor-bearing mice and the targeted enrichment in the tumor were observed. The results at 4-8 H after administration are shown in FIGS. 1A-1F. Figure 4 As shown in FIGS. 1A-1F, the results show that the fluorescent contrast agent MPA-PEG2-SLV1 (A) and MPA-PEG2-SLV2 (B) can target pancreatic cancer, liver cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer and brain glioma. Figure 4 A), SLV2( Figure 4 B) can target pancreatic cancer, liver cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer and brain glioma.
[0062] The in vitro cell imaging experiment results show that the FITC-PEG2-SLV1 and SLV2 probes obtained by replacing MPA in MPA-PEG2-SLV1 and MPA-PEG2-SLV2 with FITC can also specifically bind to the cell membrane of colorectal cancer and lung cancer cells; flow cytometry detection shows that the Kd of SLV1 and SLV2 specifically binding to colorectal cancer cells is about 5-10 μM. Figures 5-12
[0063] The above only describes the preferred embodiments of the present application, and it should be noted that those of ordinary skill in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. A cancer targeting peptide, characterized in that, The amino acid sequence is shown as SEQ ID NO. 1, SEQ ID NO.
2.
2. The cancer targeting peptide according to claim 1, characterized in that, The cancer targeting peptide is a linear peptide.
3. A polypeptide derivative, characterized in that, The polypeptide derivative is obtained by modifying PEG2 at the N-terminal and NH2 at the C-terminal of the polypeptide of claim 1.
4. A polypeptide conjugate, characterized in that, The polypeptide conjugate is obtained by coupling the polypeptide derivative of claim 3 with a carrier.
5. The polypeptide conjugate of claim 4, wherein, The carrier is selected from one of a cytokine, a radioactive element, a carrier protein, an antibody, an enzyme, a fluorescent group, a quantum dot, or a high light absorption coefficient chromophore.
6. The polypeptide conjugate of claim 5, wherein, The fluorescent group is a near-infrared region 1 fluorescent dye and / or a near-infrared region 2 fluorescent dye.
7. The polypeptide conjugate of claim 6, wherein, The near-infrared region 1 fluorescent dye includes one of MPA, IRDye800, IR820, Cy7.5, Cy7, ICG, and Cy5.5, and the near-infrared region 2 fluorescent dye includes FD1080 and / or CH1055.
8. Use of the polypeptide conjugate of any one of claims 4-7 in the preparation of a medicament for treating cancer, or in the preparation of a cancer imaging agent.
9. Use according to claim 8, characterized in that, The cancer includes, but is not limited to, pancreatic cancer, liver cancer, breast cancer, colorectal cancer, lung cancer, esophageal cancer, and brain glioma.
10. Use according to claim 8, characterized in that, The cancer imaging agent is used for precise positioning of tumor boundaries and / or intraoperative navigation fluorescence.