Short peptide targeting and recognizing neuroendocrine tumor marker INSM1 and products and applications thereof

Abstract

The application discloses a polypeptide for targeted recognition of a neuroendocrine tumor marker INSM1 and products and applications thereof, and belongs to the fields of molecular imaging and protein polypeptide drug engineering. Specifically, the application relates to development of a polypeptide with high affinity and specificity to a neuroendocrine tumor marker insulinoma-associated protein 1 (INSM1). The application discloses an amino acid sequence of the polypeptide and related applications. The polypeptide is used for labeling and preparation of a polypeptide probe, can be specifically combined with the INSM1 protein, and can be used for in-vivo and in-vitro imaging of neuroendocrine tumors, so as to overcome a diagnosis difficulty caused by a hidden onset of the neuroendocrine tumor and realize early positioning and qualitative diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of molecular imaging and protein and peptide drug engineering technology, specifically relating to a short peptide that targets and identifies the neuroendocrine tumor marker INSM1, its product (probe), and its applications. Background Technology

[0002] Neuroendocrine neoplasms (NENs) are a relatively rare type of tumor, derived from neuroendocrine cells. They can occur in various parts of the body, including the lungs, gastrointestinal tract, pancreas, pituitary gland, thyroid gland, prostate, uterus, and bones. Digestive system neuroendocrine tumors are the most common. Epidemiological analysis suggests that the incidence of neuroendocrine tumors is gradually increasing. Approximately half of gastrointestinal and pancreatic neuroendocrine tumors are malignant. Although the development of NENs is relatively slow compared to other digestive tract tumors, the invasive and metastatic characteristics of malignant tumors still indicate a high mortality rate. Some neuroendocrine tumors secrete hormones such as polypeptides and neuroamines, making them functional tumors. These tumors can exhibit specific symptoms through these biologically active secretions. However, most neuroendocrine tumors are non-functional, lacking specific symptoms and often only discovered when local tumor growth causes compressive symptoms or when distant metastasis occurs. Neuroendocrine tumors grow slowly, have low malignancy, and early resection results in a high cure rate. However, precisely because of this characteristic, most neuroendocrine tumors often lack specific clinical symptoms, especially in the early stages, and cannot be detected in time, causing most patients to lose the opportunity for a cure by the time they are diagnosed.

[0003] Currently, the main methods for screening and diagnosing neuroendocrine tumors used in clinical practice include the following: First, laboratory tests can be conducted to check endocrine tumor markers. For functional tumors, endocrine tests such as serum hormone levels and stimulation tests can also be used for screening. Second, imaging techniques such as CT, MRI, endoscopy, ultrasound, and even interventional imaging are used to visualize and locate the tumor. Third, if tumor tissue can be obtained through puncture or biopsy, pathological and immunohistochemical methods can be used to determine the diagnosis and pathological type, thereby formulating a treatment plan. However, the above three examination methods have limitations in practice. Compared with local tumor tissue, the concentration of serum tumor markers is significantly reduced, and the sensitivity and specificity are insufficient. Therefore, they are often used as auxiliary diagnostic methods and lack detection value themselves. Positive hormone tests are only seen in functional tumors with significantly increased hormone levels; however, they are often meaningless in non-functional tumors and early functional tumors. Unlike serological tests, imaging screening can clearly identify the location and size of tumors, which is significant for detecting neuroendocrine tumors. However, imaging examinations cannot characterize the tumor, i.e., they cannot determine its tissue origin or benign / malignant nature, thus failing to achieve a definitive diagnosis. A biopsy or surgery is necessary to obtain tissue for pathological diagnosis before a corresponding treatment plan can be developed. Furthermore, due to the limitations of different imaging examinations, smaller tumors may require invasive procedures such as vascular intervention or endoscopic ultrasound. Whether it's a biopsy, surgical procedure, vascular intervention, or endoscopic ultrasound, all are invasive procedures, often difficult to perform in primary healthcare institutions, placing a burden on patients' physical and mental health, finances, and social medical resources. Therefore, developing a highly sensitive, highly specific, and relatively non-invasive method for detecting neuroendocrine tumors has significant application value.

[0004] Traditional imaging relies on the examiner's morphological judgment. However, current advancements in disease-related molecular mechanisms and imaging technology allow for molecular-level imaging targeting disease-specific molecules, enabling qualitative and quantitative assessments of the molecular biological behavior of diseases through imaging. This is achieved by identifying specific ligands for disease molecular markers and binding them to detection groups (isotopes, fluoresceins, magnetic materials, acoustic contrast agents, etc.) to form molecular probes. These probes are then used for imaging using techniques such as PET-CT, endoscopy, MRI, and ultrasound. Currently, the main bottleneck restricting molecular imaging technology is the development of disease-related molecular probes. A clinically applicable molecular probe needs the following characteristics: First, specificity and affinity for diseased tissue, meaning the probe binds only to diseased tissue, not background tissue, achieving a strong signal-to-noise ratio; second, in vivo stability and suitable pharmacokinetic properties, meaning the probe can reach the lesion site before degradation, providing stable imaging, and can be rapidly cleared afterward to reduce potential side effects; third, convenient and inexpensive probe preparation. Currently, common molecular ligands include antibodies and antibody fragments, peptides, nucleic acid aptamers, and natural or synthetic small molecules. Antibodies possess the strongest specificity, but their large molecular weight, complex structure, high cost, and significant side effects limit their application. Small molecule probes are currently the most widely used due to their simple preparation and stable structure, but their poor specificity leads to suboptimal diagnostic results. Peptide probes share a similar molecular basis with antibodies, thus exhibiting better specificity. Compared to antibodies, peptides have smaller molecular weights, relatively lower in vivo immunogenicity, and can be chemically synthesized, significantly reducing preparation costs. Phage display peptide library screening technology is an important method for obtaining affinity peptides targeting specific sites. Its principle involves inserting a gene sequence library encoding a peptide library into the gene encoding the phage capsid protein. Therefore, exogenous peptides can be fused and expressed on the phage surface, enabling the use of phages to achieve 10... 9 High-throughput screening at this level. Currently, reports on phage display peptide library screening for neuroendocrine tumor markers are relatively rare.

[0005] Insulinoma-associated protein 1 (INSM1) is physiologically expressed in neuroendocrine cells during fetal development, but its expression declines after birth, making it difficult to detect in normal mature organs. In neuroendocrine tumor cells, multiple reports show high expression and high tissue specificity. INSM1 has a positive rate of over 90% in thoracic malignant neuroendocrine tumors, higher than traditional neuroendocrine tumor markers such as Syn, CgA, and CD56. INSM1 specificity is also significantly higher than traditional neuroendocrine tumor markers, reaching 99%, compared to less than 55% in lung cancer. In pancreatic neuroendocrine tumors, INSM1 expression increases with tissue grade (malignancy), but is not expressed in pancreatic cancer. In gastrointestinal neuroendocrine tumors, the expression rate is 100% in primary lesions and as high as 94% in metastatic lesions. In summary, INSM1 is highly expressed in the vast majority of neuroendocrine tumors with extremely high specificity, making it an ideal biomarker and diagnostic and therapeutic target. Specific affinity ligands developed for INSM1 can bind to neuroendocrine tumor cells and tissues via INSM1, and have the potential for targeted diagnosis and treatment of neuroendocrine tumors in clinical practice. However, there are no reports on the development of neuroendocrine tumor-targeting probes for INSM1. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide a short peptide that targets and identifies the neuroendocrine tumor marker INSM1, as well as its products and applications, to solve the current deficiencies of low serum marker positivity rate and inability to definitively identify neuroendocrine tumors by imaging examinations.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] This invention discloses a short peptide that targets and identifies INSM1, a neuroendocrine tumor marker, and the amino acid sequence of the short peptide is shown in SEQ ID No:1.

[0009] This invention discloses a fusion protein composed of a short peptide that targets and recognizes the neuroendocrine tumor marker INSM1 and a bioactive polypeptide.

[0010] Preferably, the bioactive polypeptide is a polypeptide with therapeutic activity, a polypeptide with binding activity, or a polypeptide with enzymatic activity.

[0011] The present invention also discloses a composition comprising the above-described short peptide that targets and recognizes the neuroendocrine tumor marker INSM1, or the above-described fusion protein, and at least one pharmaceutically acceptable excipient or carrier.

[0012] The present invention also discloses a probe for targeting and recognizing the neuroendocrine tumor marker INSM1, which is composed of the aforementioned short peptide for targeting and recognizing the neuroendocrine tumor marker INSM1 and an imaging group.

[0013] Preferably, the imaging group includes one or more of fluorescent dyes, chromophore dyes, chemiluminescent compounds, bioluminescent proteins, enzymes, and radionuclides.

[0014] This invention also discloses the application of the above-mentioned short peptide, fusion protein, and probe targeting the neuroendocrine tumor marker INSM1 in the preparation of diagnostic reagents for diagnosing neuroendocrine tumors.

[0015] The present invention also discloses a diagnostic kit for neuroendocrine tumors, comprising a short peptide that targets and identifies the neuroendocrine tumor marker INSM1, the amino acid sequence of which is shown in SEQ ID No:1.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention discloses a short peptide capable of targeting and recognizing the neuroendocrine tumor marker INSM1. INSM1 exhibits higher sensitivity and specificity compared to traditional neuroendocrine tumor markers such as Syn, CgA, and CD56. This short peptide consists of only 12 amino acids, has a small molecular weight, and is easy to synthesize. Using this peptide to link an imaging group, probes can be prepared. Combined with imaging techniques, this allows for targeted detection of neuroendocrine tumors in vivo, achieving non-invasive localization and qualitative examination of the tumor. It possesses good affinity and specificity. Therefore, this invention effectively overcomes the current deficiencies in the diagnosis of neuroendocrine tumors, such as low positive rates of serum markers and the inability of traditional imaging examinations to provide qualitative diagnosis. It also overcomes the diagnostic challenges caused by the insidious onset of neuroendocrine tumors, enabling early localization and qualitative diagnosis, thus possessing high clinical application value. Attached Figure Description

[0018] Figure 1 The present invention enables the polypeptide probe to bind to pancreatic neuroendocrine tumor cells BON-1 (A), normal human pancreatic duct epithelial cells HPDE6-C7 (B), and human pancreatic cancer cells PNAC1 (C), while the fluorescently labeled control peptide binds to pancreatic neuroendocrine tumor cells BON-1 (D).

[0019] Figure 2 This is the in vivo imaging result of the polypeptide probe of the present invention on the tumor in a nude mouse xenograft model;

[0020] Figure 3 This is a statistical result of the fluorescence intensity of tumors and other organs 2 hours after intravenous injection of the polypeptide probe of this invention into a nude mouse xenograft model. Detailed Implementation

[0021] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0022] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0023] The present invention will now be described in further detail with reference to the accompanying drawings:

[0024] 1. Using phage display peptide library technology and recombinant human INSM1 purified protein as the target, a solid-phase subtractive screening process was employed. After four rounds of screening, phages with affinity for the target protein were gradually enriched. After the final round of screening, affinity phages were selected for sequencing, and the sequence with the highest number of duplicate clones was chosen to obtain the affinity peptide ligand sequence WQKPSHIPFNAS (Typ-Gln-Lys-Pro-Ser-His-Ile-Pro-Phe-Asn-Ala-Ser).

[0025] 2. The short peptide sequences obtained through screening were synthesized in a solid-phase manner using an automated peptide synthesizer. Starting from the carboxyl terminus, amino acids were added one by one to the amino terminus using the Fmoc method. The specific procedure was as follows: an alkaline solvent was used to remove the protecting group of the amino group; an activator was used to activate the carboxyl group of the next amino acid, which then cross-linked with the free amino group of the previous amino acid to form a peptide bond. This cycle was repeated until the synthesis of the dodecapeptide was completed. Finally, the short peptide was eluted from the column. The short peptide was labeled with fluorescein (FITC) at its N-terminus, and FITC was linked to the naked amino group of the peptide to form a probe. Subsequent verification was conducted to validate the affinity, specificity, and imaging effect of the short peptide for neuroendocrine tumor cells.

[0026] 3. The probes prepared above were then bound to pancreatic neuroendocrine tumor cells BON-1, normal human pancreatic ductal epithelial cells HPDE6-C7, and human pancreatic cancer cells PNAC1, using the following specific method:

[0027] Cell slides were taken, fixed with 4% paraformaldehyde, blocked with 3% BSA for 30 min, then diluted peptide probe and control fluorescein-labeled peptide (10 μM) were added, incubated at 37℃ for 10 min, nuclei were stained with DAPI, mounted with glycerol, and observed under a fluorescence microscope. Figure 1 As shown, BON-1 neuroendocrine tumor cells after probe binding ( Figure 1 The middle A) showed obvious green fluorescent staining, while normal human pancreatic duct epithelial cells HPDE6-C7 ( Figure 1 (B) and human pancreatic cancer cells PNAC1 ( Figure 1 (C) showed no obvious staining. The control fluorescently labeled peptide did not bind to BON-1. Figure 1 The results (D) suggest that the probe can specifically bind to neuroendocrine tumor cells, but has no affinity for normal pancreatic cells and pancreatic cancer cells.

[0028] 4. Use animal models to verify the in vivo imaging capability of the peptide probe.

[0029] The specific method is as follows: Prepare a single-cell suspension from BON-1 cells in good growth condition. Take 6-week-old nude mice and use 2×10⁻⁶ cells... 6 0.2 mL of cells were seeded into the left axilla of nude mice to establish a subcutaneous xenograft model of human neuroendocrine tumors. After successful tumor formation, 100 μL of a 200 μM fluorescein-labeled probe and a fluorescein-labeled control peptide were injected via the tail vein from the nude mice. The mice were anesthetized with isoflurane, and the fluorescence intensity of the xenograft and other tissues and organs was observed every 60 minutes using a small animal in vivo imaging system. Figure 2 As shown, the polypeptide probe began to accumulate and visualize at the tumor site 1 hour after injection, with higher intensity than other tissues. The tumor fluorescence intensity was highest 2 hours after injection and was excreted from the bladder 6 hours later. The fluorescently labeled control peptide (FITC-labeled BON-1 homoamino acid disordered peptide) showed no tumor aggregation effect and was excreted from the bladder 2 hours after injection. Figure 3 The fluorescence intensity statistics of various organs were presented 2 hours after injection. It was found that the concentration of the BON-1 probe in tumor tissue was significantly higher than in normal tissues of the heart, lungs, liver, kidneys, pancreas, brain, and stomach, while the concentration of the fluorescently labeled control peptide showed no significant difference between tumor and normal organ tissues. This confirms that peptide probes can achieve tumor imaging in vivo with rapid clearance and low risk.

[0030] In summary, this invention has developed a short peptide with high affinity and specificity for insulinoma-associated protein 1 (INSM1), a marker of neuroendocrine tumors. Experimental verification has shown that using this short peptide for labeling and preparing short peptide probes can specifically bind to INSM1 protein and visualize neuroendocrine tumors in vitro and in vivo. This overcomes the diagnostic challenges caused by the insidious onset of neuroendocrine tumors and enables early localization and qualitative diagnosis.

[0031] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. Use of a short peptide which targets and recognizes a marker INSM1 of a pancreatic neuroendocrine tumor in the manufacture of a diagnostic reagent or a diagnostic kit for the diagnosis of a pancreatic neuroendocrine tumor, characterized in that, The amino acid sequence of the short peptide is shown as SEQ ID No:

1.

2. Use of a probe that targets and recognizes a marker INSM1 of a pancreatic neuroendocrine tumor in the manufacture of a diagnostic reagent or a diagnostic kit for the diagnosis of a pancreatic neuroendocrine tumor, characterized in that, The probe for targeting and recognizing the pancreatic neuroendocrine tumor marker INSM1 is composed of a short peptide for targeting and recognizing the pancreatic neuroendocrine tumor marker INSM1 and an imaging group; The amino acid sequence of the short peptide for targeting and recognizing the pancreatic neuroendocrine tumor marker INSM1 is shown as SEQ ID No:

1.

3. Use according to claim 2, characterized in that, The imaging group comprises one or more of a fluorescent dye, a chromophoric dye, a chemiluminescent compound, a bioluminescent protein, an enzyme and a radionuclide.

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