Directed induction of t cell lytic activity peptides, methods of making and use in treating lung nodule formulations

Abstract

The application discloses a kind of directional induction T cell lysis active peptide and its preparation method and application in treating lung nodule preparation.The active peptide of the application realizes effective intervention to the development process of lung nodule by multi-target, multi-pathway synergistic effect, i.e., inhibiting inflammatory pyroptosis, enhancing antioxidant capacity, regulating immune homeostasis and reducing potential malignant markers.Compared with the passive strategy of existing clinical "wait and observe", the application provides a solid experimental basis and a promising candidate molecule for developing a new drug capable of early and active intervention of lung nodule, and is expected to fill the gap in the field of early drug treatment of lung nodule, and has important clinical conversion value.

Description

Technical Field

[0001] This invention relates to the field of biological agents, and more particularly to a targeted T-cell lysis-inducing active peptide, its preparation method, and its application in a formulation for treating pulmonary nodules. Background Technology

[0002] Pulmonary nodules are round or oval, opaque radiographic (CT scan) shadows within the lung parenchyma, typically ≤3 cm in diameter. They are classified by size as: micronodules (<5 mm), small nodules (5-10 mm), and nodules (>10 mm). They are also classified by density as: solid nodules (completely obscuring lung tissue) and subsolid nodules (including pure ground-glass opacities (like a small patch of hazy mist) and partially solid nodules (mixed types, containing both ground-glass and solid components). Partially solid nodules have a relatively higher probability of malignancy, especially as their solid component increases over time. Finally, they are classified by nature as: benign (the vast majority): such as inflammatory pseudotumors, granulomas (tuberculosis, fungal infections), hamartomas, and lymph nodes. Malignant nodules are primarily lung cancer (adenocarcinoma is most common, especially associated with ground-glass opacities), but metastatic tumors are also possible.

[0003] Not all incidentally discovered pulmonary nodules require treatment or surgery. Management typically includes: regular CT follow-up (to observe for changes), further diagnosis (such as PET-CT, biopsy), or surgical resection (which is both a diagnostic tool and a radical treatment). Given the challenges of current medical practice—limited sensitivity in detecting small or early-stage pulmonary nodules, coupled with the high difficulty and risk of conventional surgical interventions—a passive strategy of "regular CT follow-up, waiting for the nodule to grow before surgery" has been consistently adopted clinically. However, this approach carries a significant hidden risk: during the long observation and waiting period, some nodules may have already progressed or even worsened, thus missing the optimal treatment window and affecting the patient's prognosis.

[0004] Therefore, it is imperative to break free from the current clinical predicament of "passive waiting" and actively promote the research and development of novel drug treatment strategies for pulmonary nodules. If safe, effective, and early-stage interventional drugs can be developed, enabling patients to receive non-surgical systemic treatment while the nodules are still small, it is expected to significantly inhibit nodule growth, reduce the risk of malignant transformation, and thus truly achieve early diagnosis and treatment, improving patients' quality of life and long-term outcomes. This is not only an urgent need for the development of clinical medicine but also a key direction for improving the prevention and treatment of lung cancer and saving patients' lives. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide a targeted T cell lysis-inducing active peptide, which contains a small molecule protein peptide, wherein the amino acid sequence of the small molecule protein peptide is: GPIGSIGPK.

[0006] Furthermore, the active peptide is derived from IFN-γ-induced T cell lysates.

[0007] The second objective of this invention is to provide a method for preparing a targeted induced T cell lysis active peptide, which uses IFN-γ as an inducer to induce T cell differentiation, thereby obtaining an active peptide containing a small molecule protein peptide, wherein the amino acid sequence of the small molecule protein peptide is: GPIGSIGPK.

[0008] Furthermore, the above-mentioned method for preparing the targeted induced T cell lysis active peptide includes the following steps: inducing T cells to differentiate for 24-72 hours using 10-30 ng / mL IFN-γ; collecting the induced T cells in the logarithmic growth phase, sonicating them at 4℃ and 300W for 5 min with a 3 s working time and a 6 s rest time; after sonication, centrifuging at 4℃ and 8000 rpm for 10 min, and collecting the supernatant; filtering the supernatant through a 0.22 μm filter membrane to remove cell debris, obtaining the active peptide containing a small molecule protein peptide, wherein the amino acid sequence of the small molecule protein peptide is: GPIGSIGPK.

[0009] Furthermore, the IFN-γ concentration was 20 ng / mL, and the induction time was 48 hours.

[0010] A third objective of this invention is to provide the application of the above-mentioned active peptides in the preparation of formulations for treating pulmonary nodules.

[0011] A fourth objective of this invention is to provide a formulation for treating pulmonary nodules, comprising the above-mentioned active peptide and one or more pharmaceutically acceptable excipients, carriers and / or diluents.

[0012] Compared with existing technologies, the targeted T-cell lysis-inducing active peptide and its preparation method provided by this invention have the following significant advantages and beneficial effects: 1. Effectively regulates key inflammatory and pyroptosis pathways, inhibiting nodule formation at its source.

[0013] The levels of key proteins closely related to inflammasome activation and pyroptosis (including NLRP3, ASC, Caspase-1, and RIPK3) in the lung tissue of mice in the treatment group were significantly reduced. This indicates that the active peptide can effectively inhibit the abnormal activation of the NLRP3 inflammasome, block the subsequent pyroptosis cascade, thereby alleviating excessive inflammatory damage to the immune microenvironment of lung tissue and providing a molecular basis for fundamentally curbing the occurrence and development of nodules.

[0014] 2. Significantly improves oxidative stress and immune regulation in lung tissue.

[0015] The experimental results showed that the level of reduced glutathione (GSH) in the lung tissue of mice was significantly increased after treatment, indicating that its antioxidant defense capacity was enhanced and helped to alleviate the damage of oxidative stress to lung tissue. At the same time, the downregulation of interferon regulatory factor-1 (IRF-1) and interleukin-18 (IL-18) levels further revealed the positive role of this peptide in regulating immune-related signaling pathways and balancing the release of pro-inflammatory factors.

[0016] 3. Significantly reduces serum tumor-related marker levels.

[0017] Following treatment, the levels of neuron-specific enolase (NSE) and carcinoembryonic antigen (CEA) in mouse serum were significantly reduced. This decrease in marker levels indirectly suggests that the bioactive peptide may effectively inhibit the malignant progression of pulmonary nodules through the aforementioned anti-inflammatory and immunomodulatory mechanisms.

[0018] 4. Directly improves the pathological morphology of lung tissue and effectively inhibits nodule formation.

[0019] The most direct evidence comes from histopathological observation: compared with the model control group, the lung tissue of the treated mice showed only mild thickening of some alveolar septa, and almost no obvious nodule formation in the lungs. This result directly confirms that the active peptide can effectively prevent or significantly reduce the formation of pulmonary nodules at the in vivo level, protecting the normal tissue structure of the lungs.

[0020] In summary, this invention utilizes IFN-γ-induced T-cell lysis-active peptides to effectively intervene in the development and progression of pulmonary nodules through multi-target and multi-pathway synergistic effects—namely, inhibiting inflammatory pyroptosis, enhancing antioxidant capacity, regulating immune homeostasis, and reducing potential malignant markers. Compared to the current passive "wait-and-see" strategy in clinical practice, this invention provides solid experimental evidence and highly promising candidate molecules for developing a novel drug capable of early and proactive intervention in pulmonary nodules, potentially filling a gap in the current field of early drug treatment for pulmonary nodules and possessing significant clinical translational value. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0022] Figure 1The antioxidant capacity of the directed-induced bioactive peptides provided in Example 1 of this invention was measured. T cells were induced with 10, 20, and 30 ng / mL IFN-γ for 24, 48, and 72 h, respectively, to obtain nine bioactive peptides, and their antioxidant activity was tested. In the figure, (A) T-AOC; (B) DPPH free radical scavenging capacity; (C) HRF scavenging rate. *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001.

[0023] Figure 2 This is the mass spectrum of TCP provided in Embodiment 2 of the present invention.

[0024] Figure 3 The effect of TCP on mouse lung tissue provided in Example 3 of this invention. In the figure, (A) photographs of the lungs of each group of mice and (B) HE results of mouse lung tissue.

[0025] Figure 4 The effect of TCP on mouse biochemical indicators provided in Example 3 of this invention. In the figure, (A) NSE; (B) CEA; (C) GSH. *: p<0.05, **: p<0.01; ***: p<0.001.

[0026] Figure 5 The effect of TCP on mouse lung tissue-related proteins provided in Example 3 of this invention. In the figure, (A) is a western blot band diagram; (B) is a bar chart. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. Detailed Implementation

[0027] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely 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 are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0029] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0030] The main reagents are as follows: The main reagents are as follows: Benzo[a]pyrene B(a)P Aladdin DMSO Sigma Total Antioxidant Capacity (T-AOC) Assay Kit, Wuhan Yilairuit Biotechnology Co., Ltd. DPPH Free Radical Scavenging Ability Test Kit (Wuhan Yilairuit Biotechnology Co., Ltd.) Hydroxyl radical (HRF) scavenging test kit, Wuhan Elairit Biotechnology Co., Ltd. Mouse Neural-Specific Enolase (NSE) Enzyme-Linked Immunosorbent Assay Kit (Wuhan Yilairuit Biotechnology Co., Ltd.) Mouse Carcinoembryonic Antigen-Associated Cell Adhesion Molecule 1 (CEACAM1) Enzyme-Linked Immunosorbent Assay Kit (Wuhan Yilairuit Biotechnology Co., Ltd.) Reduced glutathione (GSH) colorimetric test kit Wuhan Yilairuit Biotechnology Co., Ltd. IRF-1 Antibody Cell Signaling Technology RIP3 AntibodyCell Signaling Technology NALP12 antibody Novus Caspase-1 antibody Novus ASC antibody Immunway Proteintech IL-18 antibody GAPDH antibody Proteintech Goat anti-Mouse IgG (H+L) antibody starter PBS buffer Gibco Glycine Beijing Liuyi Instrument Factory Methanol Beijing Chemical Plant Sodium Chloride Tianjin Tianli Chemical Reagent Co., Ltd. Tris-base Beijing Liuyi Instrument Factory Changsha Bio-Boyou Biotechnology Co., Ltd. Protein Quantitative Reagent Kit Beyotime Biotechnology Co., Ltd. Twain-20 Beijing 61 Instrument Factory BSA Beijing Skim milk powder Nanjing YouNeng Biotechnology Co., Ltd. NewSemi Biotechnology Co., Ltd. Protein lysis buffer and loading solution - Xinsaimei Biotechnology Co., Ltd. PVCmECL ULtra(A)(B) Xinsaimei Biotechnology Co., Ltd. Prestained protein molecular weight marker, Thermo Scientific.

[0031] Example 1: Preparation of FN-γ-induced T cell lysis active peptides The T cells used in this invention were purchased from STEMCELL Technologies, Catalog # 200-0730.

[0032] 1.1 Directional Induction T cells were induced for 24, 48, and 72 hours using IFN-γ at concentrations of 10, 20, and 30 ng / mL, respectively, under the cell isolation and culture conditions. T cells in the logarithmic growth phase after induction were collected and lysed using low-temperature sonication (300W) for 5 min (3 s working, 6 s rest). After sonication, the cells were centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter to remove cell debris, yielding an extract of nine active peptides containing small molecule protein peptides.

[0033] 1.2 Detection of antioxidant activity of targeted induced active peptides Nine bioactive peptides obtained by inducing T cells with different concentrations of IFN-γ (10, 20, and 30 ng / mL) for 24, 48, and 72 h were analyzed using a total antioxidant capacity (T-AOC), 1,1-diphenyl-2-picrylhydrazine (DPPH) free radical scavenging capacity, and hydroxyl radical (HRF) scavenging rate assay kit (Wuhan Yilairuit Biotechnology Co., Ltd.) according to the manufacturer's instructions. T-AOC levels are expressed in mM, DPPH free radical scavenging capacity in mmol / L, and HRF scavenging rate in %. The antioxidant capacity of these nine bioactive peptides was determined by detecting their total antioxidant capacity (T-AOC), 1,1-diphenyl-2-picrylhydrazine (DPPH) free radical scavenging capacity, and hydroxyl radical (HRF) scavenging rate.

[0034] The results are as follows Figure 1 As shown, the active peptide (TCP) obtained after inducing T cells with 20 ng / mL IFN-γ for 48 h had the strongest antioxidant capacity, and we used this induction concentration and time in all subsequent experiments.

[0035] 1.3 Key Points of Total Quality Control (QC) (1) T cell stage: no mycoplasma, no viral contamination, normal karyotype, and positive rate of pluripotency markers ≥95%; (2) Active peptide stage: purity ≥98%, endotoxin content <0.1 EU / mg, no host protein residue; (3) HLCs stage: viable cell rate ≥90%, teratoma formation risk is 0, no tumorigenicity; (4) Formulation stage: Cell preparations are sterile and pyrogen-free, and the stability of active peptide preparations meets the pharmacopoeia requirements (stored at 4℃ for more than 6 months).

[0036] Three batches of active peptides were prepared, and the test results all met the quality control requirements.

[0037] Example 2: Determination of the amino acid sequence of active peptides by liquid chromatography-mass spectrometry (LC-MS / MS) 0.5 μg of active peptide (TCP) was dissolved in solution A (0.1% formic acid aqueous solution). The peptide was loaded into a 2 cm pre-packed column (100 μm inner diameter; 3 μm C18-AQ packing material; Dr Maisch) using solution A and separated using a self-made analytical column (ReproSil-Pur C18-AQ packing material; 1.9 μm; Dr Maisch) with an inner diameter of 150 μm and a length of 12 cm.

[0038] The liquid chromatography system was Ultimate 3000 (Thermo Fisher), with a liquid phase gradient of 600 nl / min for 90 min (mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile solution) (0–8 min, 6–12% B; 8–60 min, 15–32% B; 60–79 min, 32–40% B; 79–80 min, 40–95% B; 80–85 min, 95% B; 85–86 min, 95–6% B; 86–90 min, 6% B).

[0039] The mass spectrometer was an Orbitrap Q Exactive (Thermo Fisher), the mass spectrometry scanning method was positive ion scanning mode, the ion transmission tube temperature was set to 320 °C, and the spray voltage was 2.2 kV.

[0040] The mass spectrometer has a first-level full-scan detection range of 300-1,400 m / z, a resolution of 70,000, automatic gain control (AGC) of 5 e5, and a maximum injection time of 50 ms.

[0041] Secondary use only selects peptides with a charge of 2-6 for high-energy collision dissociation, with the energy level set at 27%, resolution at 17,500, automatic gain control (AGC) at 2e4, maximum injection time at 80ms, and dynamic exclusion time at 15s.

[0042] The mass spectrometer automatically performed alternating mass spectrometry (MS) processing on the active peptide TCP, and the results are as follows: Figure 2As shown, the main component (m / z 825.48) was analyzed by MS / MS and contained 7 amino acids: glycine (Gly, G), proline (Pro, P), isoleucine (Iso, I), serine (Ser, S), and lysine (Lys, K), with the amino acid sequence: GPIGSIGPK.

[0043] Example 3: Study on the therapeutic effect of active peptides on pulmonary nodules 3.1 Establishment of a B(a)P-induced mouse pulmonary nodule model Male C57BL / 6J mice, weighing approximately 15-20g, were selected as the animals. They were housed in standard separate cages with free access to food and water. The ambient temperature was maintained at 22±2℃, and the relative humidity at 60%±5%. The mice were allowed to acclimatize for one week prior to the experimental treatment, during which they were provided with 12 hours of light and 12 hours of darkness daily.

[0044] The mice were randomly divided into three groups: Control group, B(a)P group, and TCP group, with 10 mice in each group. In the B(a)P group, mice were administered B(a)P (50 mg / kg) twice weekly by gavage for 4 weeks. In the TCP group, mice were initially administered B(a)P (50 mg / kg) twice weekly by gavage for 4 weeks, and then TCP was injected daily via tail vein starting in the third week for 10 weeks. In the Control group, mice were administered olive oil twice weekly by gavage for 4 weeks, and then saline was injected daily via tail vein starting in the third week for 10 weeks. Mouse weight was measured twice weekly. After the treatment period, mice were allowed free access to water for 8 hours without food. They were then weighed, anesthetized with CO2, and blood and lung samples were obtained. Blood samples were centrifuged at 1300 rpm for 15 min to obtain serum samples, which were stored at 4°C for later use. A portion of the organs were fixed with 10% formaldehyde, and another portion was stored at -80°C.

[0045] 3.2 Histopathological and morphological observation HE staining: Drying and dewaxing: Place the slides in a drying machine at 60℃ for 2 hours. Then, place the slides in xylene I, II, and III for 10 minutes each, anhydrous ethanol I, II, and III for 5 minutes each, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, 75% ethanol, and wash with distilled water.

[0046] Hematoxylin staining of cell nuclei: Immerse the sections in hematoxylin for 3-8 min, then wash with tap water to restore blue color.

[0047] Eosin staining of cytoplasm: Immerse the sections in eosin staining solution for 1-3 min.

[0048] Dehydration and clearing: Quickly pass the sections through 85% alcohol, 95% alcohol for 2 seconds, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 10 minutes, and xylene II for 10 minutes to clear them.

[0049] Mounting: Add an appropriate amount of neutral resin to the slide and mount it.

[0050] Results of mouse lung photographs Figure 3 As shown in Figure 3(A), pulmonary nodules were observed at the locations indicated by the black arrows in the B(a)P group. The Control and TCP groups showed almost no pulmonary nodules. The HE results of mouse lung tissue are shown in Figure 3(B). Compared to the Control group, the B(a)P group showed significantly thickened alveolar septa with inflammatory cell infiltration (black arrow locations). Compared to the B(a)P group, the TCP group showed slight thickening of some alveolar septa.

[0051] 3.3 Enzyme-linked immunosorbent assay (ELISA) of mouse neural-specific enolase (NSE) and mouse carcinoembryonic antigen-associated cell adhesion molecule 1 (CEA) The ELISA kit was used to detect the levels of NSE and CEA in mouse serum.

[0052] Mouse serum NSE and CEA results are as follows Figure 4 As shown in (A) and (B), compared with the Control group, the levels of NSE and CEA in the B(a)P group were significantly increased (p<0.01 and p<0.05). Compared with the B(a)P group, the levels of NSE and CEA in the TCP group mice were significantly decreased (p<0.05).

[0053] 3.4 Determination of GSH content in mouse lung tissue Glutathione (GSH) levels in lung tissue samples were detected using a kit. Specifically, collected lung tissue samples were rapidly frozen in liquid nitrogen and stored at -80°C for later use. For testing, approximately 50 mg of tissue sample was homogenized in pre-chilled PBS buffer (pH 7.4), centrifuged at 12,000 × g for 15 minutes at 4°C, and the supernatant was used for subsequent analysis. 30 μL of the tissue homogenate supernatant or standard was added to a 96-well plate, followed by 120 μL of detection buffer, 50 μL of reaction mixture (containing DTNB and glutathione reductase), and 50 μL of NADPH solution. After thorough mixing, the absorbance at 412 nm was immediately monitored using a microplate reader. The GSH concentration in the sample was calculated based on the GSH standard curve, and the final result was expressed as nmol / mg protein.

[0054] The results are as follows Figure 4As shown in (C), compared with the Control group, the GSH level in the B(a)P group was significantly lower (p<0.05). Compared with the B(a)P group, the GSH level in the TCP group mice was higher.

[0055] 3.5 Changes in the level of related proteins in mouse lung tissue Western blot experiment: Sample preparation: Take hippocampal tissue, add RIPA lysis buffer (containing 1% PMSF, 1% protease inhibitor, and 1% phosphatase inhibitor), homogenize, and lyse on ice for 30 minutes. Centrifuge at 12000×g for 15 minutes at 4°C, and collect the supernatant. Determine protein concentration using the BCA method and adjust to the same concentration.

[0056] Electrophoresis and membrane transfer: Sample loading: 20-30 μg protein per well, denature at 95°C for 5 minutes.

[0057] SDS-PAGE electrophoresis: stacking gel 80V, separating gel 120V.

[0058] Transfer: Wet transfer method, 300 mA constant current, 90 minutes (PVDF membrane, 0.22 μm, activated with methanol).

[0059] Sealing: 5% skim milk powder (TBST formulation) at room temperature for 1 hour.

[0060] Incubate antibodies: Incubate the primary antibody overnight at 4°C (or for 2 hours at room temperature).

[0061] Wash TBST 3 times, 5 minutes each time.

[0062] HRP-labeled secondary antibody (CST, 1:5000) at room temperature for 1 hour.

[0063] Wash TBST 3 times, 5 minutes each time.

[0064] development: ECL chemiluminescent liquid, detected by chemiluminescence imaging systems (such as Bio-Rad ChemiDoc).

[0065] ImageJ software was used to quantitatively analyze the gray values ​​of the bands, and the target protein / internal reference ratio represents the relative expression level.

[0066] Results of changes in lung tissue-related protein levels as follows Figure 5As shown in (A) and (B), compared with the Control group, the expression of NLRP3, RIPK3, Caspase-1, IRF-1, ASC, and IL-18 proteins in lung tissue was significantly increased in the B(a)P group (p<0.01, p<0.001, and p<0.0001), indicating an inflammatory response. Compared with the B(a)P group, the expression of the above proteins was significantly decreased in the TCP group (p<0.05, p<0.001, and p<0.0001), indicating that TCP can exert its function by inhibiting inflammation-related proteins.

[0067] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A peptide that induces T cell lysis, characterized in that, It contains a small molecule protein peptide, the amino acid sequence of which is: GPIGSIGPK.

2. The targeted T cell lysis-inducing active peptide as described in claim 1, characterized in that, The active peptide was derived from IFN-γ-induced T cell lysates.

3. A method for preparing a targeted T cell lysis-inducing active peptide, characterized in that, T cells were induced to differentiate using IFN-γ as an inducer to obtain an active peptide containing a small molecule protein peptide. The amino acid sequence of the small molecule protein peptide is: GPIGSIGPK.

4. The method for preparing the targeted T cell lysis-inducing active peptide as described in claim 3, characterized in that, The procedure includes the following steps: T cells are induced to differentiate at 10-30 ng / mL IFN-γ for 24-72 hours; T cells in the logarithmic growth phase after induction are collected and sonicated at 4℃ and 300W for 5 min with a 3 s working time and a 6 s rest time; after sonication, the cells are centrifuged at 4℃ and 8000 rpm for 10 min, and the supernatant is collected; the supernatant is filtered through a 0.22 μm filter membrane to remove cell debris, yielding an active peptide containing a small molecule protein peptide, the amino acid sequence of which is: GPIGSIGPK.

5. The method for preparing the targeted T cell lysis-inducing active peptide as described in claim 3, characterized in that, The IFN-γ concentration was 20 ng / mL, and the induction time was 48 hours.

6. The use of the active peptide according to claim 1 or 2 in the preparation of a formulation for treating pulmonary nodules.

7. A preparation for treating pulmonary nodules, characterized in that, It comprises the active peptide as described in claim 1 or 2 and one or more pharmaceutically acceptable excipients, carriers and / or diluents.

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