Trop2 targeting peptide and targeting vector constructed by Trop2 targeting peptide
By expressing Trop2-targeting peptide on the exosome membrane, targeting vectors are constructed and chemotherapy drugs are loaded, the problem of insufficient exosome targeting is solved, efficient targeted treatment for breast cancer is achieved and drug toxicity is reduced.
Patent Information
- Application Number
- CN202510167913.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-17
- Publication Date
- 2025-06-03
AI Technical Summary
The prior art is difficult to achieve precise targeted tumor therapy. Exosomes are insufficiently targeted as drug carriers, resulting in uneven distribution of drugs in the body, reducing the benefits of chemotherapy and increasing toxic side effects.
Trop2-targeted peptides were screened through phage display technology and modified onto the exosome membrane to construct a Trop2-targeted vector, load the anti-breast cancer chemotherapy drug paclitaxel, and form a Trop2-targeted drug-loading complex.
It significantly enhances the targeting of exosomes, improves the inhibitory effect on breast cancer, and reduces the toxic side effects of the drug.
Smart Images
Figure CN120081903A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of breast cancer treatment, and particularly relates to a Trop2-targeted peptide and a targeted vector constructed therefrom. Background Art
[0002] Chemotherapy is one of the important means in clinical tumor treatment. However, due to the non-specific distribution of drugs in the human body and their own toxicity, it has become a difficult problem to maintain a high level of drug concentration at the tumor site. At the same time, continuous low-sensitivity chemotherapy is likely to cause drug tolerance in tumor tissues, reduce the clinical benefit brought by chemotherapy, and increase the risk of recurrence and metastasis. Therefore, the main breakthrough in achieving precise tumor-targeted therapy lies in precisely delivering chemotherapy drugs, improving the sensitivity of tumors to chemotherapy drugs, and at the same time, if real-time and effective monitoring can be carried out during chemotherapy, it will also increase the benefit of patients.
[0003] Exosomes (Exo) are a widely studied natural-source drug carrier. The microvesicle structure of exosomes (with a diameter of about 30 - 150 nm) contains a variety of biological information substances, including lipids, proteins, mRNA, microRNA, etc. It is one of the important media for intercellular material exchange and is involved in various physiological and pathological processes of the body. Compared with artificially synthesized nanomaterial carriers, exosomes of natural origin are not easily cleared by the reticuloendothelial system and have a longer retention time in the body's circulatory system.
[0004] Therefore, exosomes have better biocompatibility. In addition, exosomes also have a nanoscale size and considerable tissue permeability. However, as a carrier for small molecule drugs and indicators, the tissue targeting of exosomes still has defects. Some studies have used the specific ligands naturally present on the surface of exosomes to target specific tissues, such as using tumor-derived exosomes to target tumor tissues, but it has the risk of promoting tumor development and cannot meet the requirements of clinical applications. Many studies have tried to modify the exosome membrane to enhance the targeting to the tumor site, such as connecting some targeting peptide segments, antibodies, and aptamers, etc. For example, exosomes modified with the polypeptide GEll can targetedly transport let-7a miRNA into breast cancer cells expressing epidermal growth factor receptor, thereby inhibiting tumors.
[0005] However, these studies still have not completely solved the problem of poor exosome targeting and are difficult to achieve breakthroughs in clinical research. In addition, many of the targeting peptide segments or aptamers used in the studies are specific to a certain cell line or a certain type of tumor, and there are heterogeneities between different tumors and even within tumors. Such exosome carriers are obviously not suitable for large-scale clinical applications. Therefore, it is of great significance to develop exosome vesicles that target tumors generally.
[0006] Trophoblast cell surface antigen 2 (Trop2), also known as membrane component surface marker-1, epithelial glycoprotein-1, gastrointestinal antigen 733-1, or tumor-associated calcium signal transducer-2, is a protein encoded by the single gene TACSTD2. Trop2 maintains relatively low expression in normal tissues such as lung tissue, ovarian tissue, kidney tissue, and testis, and plays an important role in the development of various organs during embryogenesis.
[0007] In recent years, studies have found that Trop2 shows tissue-specific high expression in multiple tumor types, such as more than a dozen common tumors including digestive tract tumors (esophageal squamous cell carcinoma, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer), female reproductive system tumors (ovarian cancer, breast cancer, cervical cancer), thyroid cancer, osteosarcoma, bladder cancer, lung cancer, prostate cancer, and lymphoma. At the same time, the role of Trop2 in tumorigenesis and progression is evolutionarily conserved. A large number of studies have shown that it regulates tumor cell proliferation, metastasis, and invasion by participating in signaling pathways such as the ERK / MEK signaling pathway, NF-κB signaling pathway, cell cycle signaling pathway, Wnt signaling pathway, and Ras.
[0008] Recently, preclinical and clinical studies targeting Trop2 have been widely carried out. Mangino et al. killed human breast cancer cells (MCF7) by preparing Trop2-directed cytotoxic T lymphocytes. Lin et al. demonstrated that in a xenograft mouse model of triple-negative breast cancer cells (MDA-MB-231), Trop2-targeted fragments inhibited tumor growth. With the progress of research, antibody-drug conjugates (ADCs) targeting Trop2 have become a research hotspot. Goldenberg et al. prepared IMMU-132 (the metabolite SN-38 of irinotecan combined with Anti-Trop2), a Trop2-targeted chemotherapeutic drug. In a xenograft mouse model, the study showed that IMMU-132 could deliver SN-38 to tumor tissues more efficiently than irinotecan alone. At the same time, studies have demonstrated that humanized anti-Anti-Trop2-SN-38 conjugates can provide significant and specific anti-cancer effects in xenograft models of several tumor cells. A special material targeting Trop2 was constructed, which could achieve tumor imaging and tracing in a mouse model of prostate cancer. In clinical studies, Bardia et al. showed that in 69 patients with triple-negative breast cancer, the clinical benefit rate of IMMU-132 was 46%. Faltas et al. reported six patients with metastatic platinum-resistant urothelial carcinoma treated with IMMU-132, and 50% of the patients showed significant therapeutic effects. Heist et al. evaluated IMMU-132 in 54 treated patients with metastatic non-small cell lung cancer and demonstrated a clinical benefit rate of 43%. The above clinical studies indicate that ADC drugs targeting Trop2 have certain efficacy in the treatment of various advanced tumors, but the biological toxicity brought by ADC structural drugs still cannot be ignored. Summary of the Invention
[0009] The object of the present invention is to provide a Trop2-targeted peptide and a targeted vector constructed therefrom. The Trop2-targeted peptide has good targeting properties. The constructed targeted vector expresses the Trop2-targeted peptide on the exosome membrane, effectively enhancing the targeting of exosomes. After loading drugs, it can effectively increase the inhibitory effect on breast cancer while reducing the drug side effects.
[0010] The technical solution adopted by the present invention to solve its technical problems is: A Trop2-targeted peptide with the amino acid sequence: WPLRSKA. It is obtained by screening through phage display technology.
[0011] A Trop2-targeted vector is obtained by modifying the above-mentioned Trop2-targeted peptide onto exosomes.
[0012] The exosomes are derived from 293T cells.
[0013] A Trop2-targeted drug-loaded complex is formed by loading a chemotherapeutic drug against breast cancer onto the Trop2-targeted vector.
[0014] The chemotherapeutic drug against breast cancer is paclitaxel.
[0015] The construction method is as follows: The Trop2-targeted vector and the chemotherapeutic drug against breast cancer are co-incubated at 4 °C for 24 h.
[0016] The beneficial effects of the present invention are as follows: The Trop2-targeted peptide has good targeting property. The constructed targeted vector expresses the Trop2-targeted peptide on the exosome membrane, effectively enhancing the targeting property of the exosomes. After loading the drug, it can effectively increase the inhibitory effect on breast cancer and at the same time reduce the drug toxicity and side effects. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 It is a schematic diagram of screening Trop2-targeted peptides by phage display technology.
[0018] Figure 2 It is a graph of the cellular uptake efficiency of the Trop2-targeted drug-loaded complex.
[0019] Figure 3 It is a graph of the in vitro cytotoxic effect of the Trop2-targeted drug-loaded complex.
[0020] Figure 4 It is a graph of the results of the in vitro inhibition of tumor cell proliferation by the Trop2-targeted drug-loaded complex.
[0021] Figure 5 It is a graph of the in vivo inhibition of tumors by the Trop2-targeted drug-loaded complex. DETAILED DESCRIPTION OF THE INVENTION
[0022] The technical solutions of the present invention will be further specifically described below through specific examples.
[0023] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following examples are conventional methods in the art unless otherwise specified.
[0024] Example 1: Screening of Trop2-targeted peptides Trop2-specific binding heptapeptides were screened by in vitro binding of Trop2-conjugated magnetic beads through phage display technology (M13 phage library) for subsequent verification experiments ( Figure 1 ). The amino acid sequence of the finally screened Trop2-targeted peptide is: WPLRSKA (SEQ ID No.1).
[0025] Trop-2 Targeted Vector Targets Trop2-Highly Expressed Cells In Vitro Construct an exosome vector modified with trop2 targeting peptide (Trop2 target-peptide @membrane), and co-incubate and load paclitaxel (PTX) under the condition of 4°C / 24h to form Trop2 target-peptide @PTX drug-loaded complex. In in vitro experiments, through cell uptake (loading the GFP overexpression plasmid Trop2 target-peptide @GFP-plasmid), it was observed that: in Trop2-lowly expressed cells, the transfection efficiency of Trop2 target-peptide @GFP-plasmid was low, and the fluorescence intensity was low (green). In Trop2-highly expressed cells, the transfection efficiency of Trop2 target-peptide @GFP-plasmid was high, and the fluorescence intensity was high (green). At the same time, using the lip3000 transfection system as a positive control, it was found that the transfection efficiency of Trop2 target-peptide @GFP-plasmid was not inferior to that of the lip3000 transfection efficiency ( Figure 2 ).
[0026] Killing Effect of Trop2 Targeted Drug-Loaded Complex (Trop2@PTX) on Breast Cancer Cells In in vitro experiments, after culturing breast cancer cells (trop2 overexpressing cell line MBA-MD-231) in a 6-well plate for 24h, different drugs were added for 36h (PBS: phosphate buffered saline; Particle trop2 : Trop2 target-peptide @membrane; PTX: paclitaxel; Particle trop2 @PTX: Trop2 target-peptide @PTX), and then flow cytometry was used to detect cell apoptosis. It was found that Trop2 target-peptide @PTX) significantly increased the apoptosis rate of cells compared with the PTX or Trop2 target-peptide @membrane group (35.7% vs 22.62% / 14.05%) ( Figure 3 ).
[0027] Inhibition of Trop2 Targeted Drug-Loaded Complex (Trop2@PTX) on the Proliferation of Breast Cancer Cells In in vitro experiments, after culturing breast cancer cells (trop2 overexpressing cell line MBA-MD-231) in a 6-well plate for 24h, different drugs were added for 36h (PBS: phosphate buffered saline; Particle trop2 : Trop2 target-peptide @membrane; PTX: paclitaxel; Particletrop2 @PTX: Trop2 target-peptide @PTX), and then the cell proliferation was detected by Edu assay. It was found that Trop2 target-peptide @PTX) significantly increased the cell proliferation rate compared with PTX or Trop2 target-peptide @membrane group (yellow indicates the cell proliferation state) ( Figure 4 ).
[0028] Verification of Trop2-targeted drug-loaded complex (Trop2@PTX) in vivo tumor model MDA-MB-231 triple-negative breast cancer cells (overexpressing Trop2) were used to establish a subcutaneous tumor implantation model (subcutaneous tumor burden model) in 4-6-week-old nude mice. When the subcutaneous tumor reached 0.5 cm (diameter), drugs were injected via the tail vein once every 2 days for 6 consecutive times. Then, the tumor tissues were collected after 4-6 weeks. The experiment found that Trop2 target -peptide @PTX significantly reduced the tumor volume compared with PTX and Trop2 target-peptide @membrane ( Figure 5 ).
[0029] The above-described embodiments are only a preferred solution of the present invention and do not impose any form of limitation on the present invention. There are other variations and modifications without exceeding the technical solutions recorded in the claims.
Claims
1. A Trop2 targeting peptide, characterized in that The amino acid sequence is: WPLRSKA.
2. The Trop2 targeting peptide according to claim 1, characterized in that It is obtained by screening through phage display technology.
3. A Trop2 targeting vector, characterized in that: The method is obtained by modifying the Trop2 targeting peptide described in claim 1 onto exosomes.
4. The Trop2 targeting vector according to claim 3, characterized in that The exosomes are exosomes derived from 293T cells.
5. A Trop2 targeted drug-loaded complex, characterized in that: The method is formed by loading the anti-breast cancer chemotherapy drug onto the Trop2 targeting carrier according to claim 3.
6. The Trop2 targeted drug carrier complex according to claim 5, characterized in that: The anti-breast cancer chemotherapy drug is paclitaxel.
7. The Trop2 targeted drug carrier complex according to claim 5, characterized in that: The construction method is: incubate the Trop2 targeting vector and the anti-breast cancer chemotherapy drug at 4°C for 24 hours.