EGFR (epidermal growth factor receptor) targeted nano-vesicle combination system and application thereof

Abstract

The invention discloses an EGFR (Epidermal Growth Factor Receptor) targeted nano-vesicle combined system and application thereof in the field of non-small cell lung cancer treatment reagents, and aims to solve the problems of strong single drug resistance, poor drug targeting, immune escape and the like in KRAS mutant NSCLC treatment in the prior art. The nano vesicle comprises a nano vesicle unit and an anti-PD-L1 antibody, the nano-vesicle unit comprises a hybrid nano-vesicle, an EGFR (epidermal growth factor receptor) specific nano-antibody and a KRAS specific inhibitor; the invention is suitable for KRAS mutation non-small cell lung cancer, and can achieve the effects of significantly weakening the single drug resistance, improving the drug targeting and inhibiting immune escape.

Description

Technical Field

[0001] This invention relates to the field of therapeutic agents for non-small cell lung cancer, and more particularly to an EGFR-targeted nanovesicle combined system and its application, wherein the EGFR-targeted nanovesicle combined system can be applied to KRAS-mutant non-small cell lung cancer. Background Technology

[0002] KRAS (murine sarcoma virus oncogene) mutation is one of the most common oncogenic drivers in non-small cell lung cancer (NSCLC), especially KRAS mutations. G12D Mutation is representative of KRAS G12D Patients with KRAS mutations have a poor prognosis and a high rate of resistance to existing targeted and immunotherapies. However, currently there are limited effective targeted therapies for KRAS mutations, and clinically used KRAS inhibitors suffer from acquired resistance, poor drug targeting, and can also cause problems such as immune escape.

[0003] Studies have shown that EGFR (a member of the HER family of epidermal growth factor receptors) phosphorylation plays a crucial role in KRAS inhibitor resistance. KRAS inhibitors often induce upregulation of EGFR phosphorylation, activating downstream signaling pathways and leading to resistance. Strategies that inhibit KRAS or EGFR alone have limited efficacy, necessitating the development of a highly effective combination therapy platform that can simultaneously block the KRAS signaling pathway and inhibit EGFR activation to overcome resistance and enhance anti-tumor efficacy. Summary of the Invention

[0004] The purpose of this invention is to provide an EGFR-targeting nanovesicle combination system and its application in the preparation of nanovesicle units, overcoming the problems of strong single-drug resistance, poor drug targeting, and immune escape in the existing treatment of KRAS-mutant NSCLC, so as to achieve KRAS-targeting... G12D Highly efficient synergistic inhibition of mutant tumors.

[0005] To achieve the above objectives, the present invention is implemented using the following technical solution:

[0006] On the one hand, the present invention provides an EGFR-targeted nanovesicle combined system, characterized in that it includes nanovesicle units and immune checkpoint inhibitors;

[0007] The nanovesicle unit includes hybrid nanovesicles, EGFR-specific nanobodies, and KRAS-specific inhibitors.

[0008] In the above technical solution, EGFR-specific nanobody (EGa1) modification is used to achieve high specificity recognition and enrichment of KRAS mutant NSCLC cells, significantly improve the drug delivery efficiency in tumor tissue, and enhance the targeting of the EGFR-targeted nanovesicle combined system.

[0009] The delivery system formed by nanovesicle units can effectively reduce the systemic toxicity of KRAS-specific inhibitors, reduce non-specific drug distribution in normal tissues, and improve safety.

[0010] The EGFR-targeted nanovesicle combination system, in which nanovesicle units and immune checkpoint inhibitors are used in synergy, can simultaneously inhibit the EGFR and KRAS signaling pathways, block drug resistance-related feedback loops, and synergistically block immune blockade by immune checkpoint inhibitors, thereby significantly improving the sensitivity of tumors to radiotherapy and drug therapy.

[0011] The EGFR-targeted nanovesicle combined system, as a combined immunoassay agent, can reverse the KRAS tumor-associated immunosuppressive microenvironment, promote T cell infiltration and activation, and enhance anti-tumor immune response;

[0012] The EGFR-targeted nanovesicle combined system has significant in vivo efficacy: the EGFR-targeted nanovesicle combined system can significantly inhibit tumor growth and lung metastasis, prolong animal survival, and has good prospects for translational application.

[0013] Furthermore, the hybrid nanovesicles are membrane-mimicking structures, formed by the co-assembly of synthetic lipid components and natural cell membrane-derived components;

[0014] The EGFR-specific nanobody is covalently or coupled to the surface of the hybrid nanovesicles;

[0015] The KRAS-specific inhibitor is encapsulated within the vesicles of the hybrid nanovesicles.

[0016] In the above technical solutions, membrane-inspired hybrid nanovesicles (hNVs) are a nanomedicine delivery system. Through targeted design, the hybrid nanovesicles are modified with specific ligands to improve targeting, prolong in vivo circulation time, and reduce immune clearance. EGa1 is covalently or coupled to the surface of the hybrid nanovesicles to achieve active targeting of EGFR-highly expressed tumor cells. KRAS-specific inhibitors are delivered in a sustained manner through encapsulation within the hybrid nanovesicles, enabling efficient drug delivery and regulation of the tumor microenvironment.

[0017] In this scheme, the hybrid nanovesicles are co-assembled from synthetic lipid components and natural cell membrane-derived components. The natural targeting of the biomembrane and the high encapsulation rate and controllable release characteristics of the nanocarrier enable the drugs contained therein to significantly inhibit tumor growth and induce apoptosis, thereby enhancing the anti-tumor immune response.

[0018] Furthermore, the KRAS-specific inhibitor includes KRAS. G12D Specific inhibitors;

[0019] The KRAS G12D Specific inhibitors include MRTX1133;

[0020] The immune checkpoint inhibitors include anti-PD-L1 antibodies;

[0021] The anti-PD-L1 antibody includes InVivoMAb anti-mouse PD-L1 antibody.

[0022] In the above technical solution, KRAS is delivered via membrane-inspired hybrid nanovesicles. G12D Specific inhibitors can achieve KRAS G12D Highly efficient synergistic inhibition of mutant tumors;

[0023] The nanovesicle units encapsulating MRTX1133 within the hybrid nanovesicles are called the EGa1-hNVs@MRTX1133 system (abbreviated as EGa1-hNVs-M). Administered via tail vein injection, it selectively accumulates in KRAS in vivo. G12D The drug is continuously released into NSCLC tumor tissue and within the tumor microenvironment, increasing the local effective concentration of KRAS inhibitors and reducing exposure to normal tissues.

[0024] The EGa1-hNVs-M synergistic anti-PD-L1 antibody (αPD-L1) used in combination with EGa1-hNVs-MP can simultaneously inhibit EGFR feedback activation and PD-L1-mediated immunosuppression, thereby significantly enhancing the anti-tumor effect of MRTX1133.

[0025] The nanovesicle units in EGa1-hNVs-MP are used in combination with anti-PD-L1 antibodies (αPD-L1 antibodies) to inhibit the KRAS / EGFR / PD-L1 immune escape axis.

[0026] Furthermore, the preparation method of the nanovesicle unit includes the following steps:

[0027] Preparation of cell membrane solutions expressing EGFR-specific nanobodies;

[0028] Prepare liposome solutions containing KRAS-specific inhibitors;

[0029] Cell membrane solution and liposome solution are mixed and extruded through a nanofiltration membrane to form nanovesicle units.

[0030] Furthermore, it also includes the following steps:

[0031] After forming nanovesicle units, the free drug is removed by centrifugation through a 50-150 kDa ultrafiltration tube.

[0032] Furthermore, the preparation of the cell membrane solution includes the following steps:

[0033] Using a vector containing EGFR-specific nanobodies, tool cells were transfected with lentiviruses, and cell lines that stably expressed EGFR-specific nanobodies were screened to obtain cell lines. Cell lines that stably expressed EGFR-specific nanobodies were collected, suspended in Tris buffer, sonicated, centrifuged to remove organelle residues, and then centrifuged again to obtain purified cell membranes, thus obtaining cell membrane solutions expressing EGFR-specific nanobodies.

[0034] The preparation of the liposome solution includes the following steps:

[0035] Synthetic phospholipids, phospholipid-polymer conjugates, and cholesterol were dissolved, and a KRAS-specific inhibitor was added. The mixture was then rotary evaporated to form a film. After vacuum drying to remove the solvent, the film was resuspended in phosphate buffer solution and sonicated to form a liposome solution containing the KRAS-specific inhibitor.

[0036] In the above technical solutions, the preparation process of nanovesicle units is simple and highly scalable, suitable for the combined delivery of various small molecule, nucleic acid and protein drugs, and has the potential for platform-based development.

[0037] Furthermore, the ratio of the cell membrane solution to the liposome solution is 1:(3~12) by mass.

[0038] The nanofiltration membrane extrusion includes the following steps: extruding nanofiltration membranes of 400 nm, 200 nm and 100 nm sequentially 10 to 20 times respectively;

[0039] The nanofiltration membrane includes a polycarbonate membrane.

[0040] Furthermore, the carrier containing the EGFR-specific nanobody includes the pCDH-CMV-EGa1-VHH-Myc-PDGFRβTM-Puro carrier;

[0041] The tool cells include 293T cells;

[0042] The process of screening to obtain a cell line that stably expresses EGFR-specific nanobodies includes the following steps: Western blot and immunofluorescence detection showed that the Myc tag signal indicated that the EGFR-specific nanobodies were successfully localized on the cell membrane surface, which is the cell line that stably expresses EGFR-specific nanobodies.

[0043] The Tris buffer contains potassium chloride, magnesium chloride, Tris, and a protease inhibitor.

[0044] The molar ratio of potassium chloride, magnesium chloride, and Tris in the Tris buffer solution is (3~7):1:(8~12).

[0045] The volume ratio of the protease inhibitor to the cell membrane solution is (80~120):1;

[0046] The ultrasonic fragmentation temperature is 2~6℃, and the time is 8~12 minutes;

[0047] The centrifugation speed for removing organelle residues is 8000~12000×g, and the centrifugation time is 8~12 minutes;

[0048] The centrifugation speed for obtaining the purified cell membrane after further centrifugation is 80,000~120,000×g, and the centrifugation time is 0.5~1h.

[0049] Furthermore, the synthetic phospholipid includes DPPC;

[0050] The phospholipid-polymer conjugate includes DSPE-PEG5K;

[0051] The molar ratio of the synthetic phospholipid, phospholipid-polymer conjugate, and cholesterol is (1-5):(1-3):1;

[0052] The solvents used to synthesize phospholipids, phospholipid-polymer conjugates, and cholesterol include chloroform;

[0053] The mass ratio of the KRAS-specific inhibitor to the liposome is (0.5~6mg):6mg;

[0054] The step of removing solvent by vacuum drying includes:

[0055] Freeze-dry at -70~-90℃ for 2~5 hours;

[0056] Vacuum freezing at -20~-25℃ for 6~10 hours.

[0057] On the other hand, the present invention provides an application of an EGFR-targeted nanovesicle combination system in the preparation of a combination therapy agent for KRAS-mutant non-small cell lung cancer.

[0058] Compared with existing technologies, this invention uses EGFR-targeting nanovesicle units to encapsulate KRAS-specific inhibitors and synergizes with anti-PD-L1 antibodies, achieving the following beneficial effects:

[0059] By modifying with EGFR-specific nanobodies (EGa1), we can achieve highly specific recognition and enrichment of KRAS mutant NSCLC cells, significantly improve the drug delivery efficiency in tumor tissues, and enhance the targeting of the EGFR-targeted nanovesicle combined system.

[0060] The delivery system formed by nanovesicle units can effectively reduce the systemic toxicity of KRAS-specific inhibitors, reduce non-specific drug distribution in normal tissues, and improve safety.

[0061] The synergistic use of nanovesicle units and anti-PD-L1 antibodies in the EGFR-targeted nanovesicle combination system can significantly improve the sensitivity of tumors to radiotherapy and drug treatment by simultaneously inhibiting the EGFR and KRAS signaling pathways, blocking drug resistance-related feedback loops, and synergistically blocking the immune response of anti-PD-L1 antibodies.

[0062] EGFR-targeted nanovesicle combination system as a combined immunoassay agent can reverse KRAS The tumor-associated immunosuppressive microenvironment promotes T cell infiltration and activation, enhancing the anti-tumor immune response;

[0063] The EGFR-targeted nanovesicle combined system has significant in vivo efficacy: the EGFR-targeted nanovesicle combined system can significantly inhibit tumor growth and lung metastasis, prolong animal survival, and has good prospects for translational application.

[0064] The EGFR-targeted nanovesicle combination system, applied as a combination therapy for KRAS-mutant non-small cell lung cancer, can significantly reduce problems such as strong single-drug resistance, improve drug targeting, and inhibit immune escape. Attached Figure Description

[0065] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0066] Figure 1 These are schematic diagrams illustrating the construction process and principle of the nanovesicle combined system and mouse experiments provided by some embodiments of the present invention;

[0067] Figure 2 This is a schematic diagram of the characterization results of EGa1-hNVs-M according to some embodiments provided by the present invention;

[0068] Figure 3 This is a schematic diagram showing the cytotoxicity and signaling pathway changes of EGa1-hNVs-M in some embodiments provided by the present invention;

[0069] Figure 4 This is a schematic diagram illustrating the in vivo targeting and biodistribution evaluation results of EGa1-hNVs-M, including some embodiments provided by this invention, in mice;

[0070] Figure 5This is a schematic diagram of the anti-tumor comparative experimental results of mouse subcutaneous xenograft tumor models, including EGa1-hNVs-M, according to some embodiments provided by the present invention;

[0071] Figure 6 This is a schematic diagram illustrating the comparative evaluation results of mouse lung metastasis models according to some embodiments of the present invention;

[0072] Figure 7 This invention provides some embodiments of spontaneous KRAS. G12D A schematic diagram illustrating the comparative validation results of the mutant lung cancer model. Detailed Implementation

[0073] A specific embodiment of the present invention provides an EGFR-targeting nanovesicle combination system, referred to as EGa1-hNVs-MP, for overcoming drug resistance in KRAS-mutant non-small cell lung cancer. This system consists of EGa1-hNVs-M and an anti-PD-L1 antibody, which are used synergistically. EGa1-hNVs-M comprises hybrid nanovesicles (hNVs) formed by the fusion of an EGa1-expressing cell membrane and a drug-loaded liposome. The drug loaded within the vesicles is KRAS. G12D The inhibitor MRTX1133. Enhanced anti-tumor efficacy is achieved through combined tumor targeting mediated by EGa1 ligand and immune checkpoint blockade. The construction and mouse experimental procedures and principles of the nanovesicle combined system are as follows: Figure 1 As shown.

[0074] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use.

[0075] Example 1

[0076] This embodiment provides the preparation and characterization of EGa1-hNVs-M.

[0077] Construction of EGa1-293T cells:

[0078] Using the pCDH-CMV-EGa1-VHH-Myc-PDGFRβTM-Puro vector, 293T cells were transfected with lentivirus, and cell lines stably expressing EGa1 were obtained through screening. Western blot and immunofluorescence assays showed that EGa1 was successfully localized to the cell membrane surface using the Myc tag signal, identifying these as EGa1-293T cells.

[0079] Extraction of EGa1 cell membrane:

[0080] EGa1-293T cells were collected and suspended in Tris buffer (10 mM KCl, 2 mM MgCl2, 20 mM Tris, with 1% 100× protease inhibitor added, the volume ratio of protease inhibitor to cell membrane being 100:1). The protease inhibitor was obtained from Suzhou Genewiz Biotechnology Co., Ltd., order number 80-1366892214. After sonication at 4°C for 10 minutes (60% power, start for 2 seconds, pause for 3 seconds), the cells were centrifuged at 10000×g for 10 minutes to remove organelle residues, and then centrifuged at 100000×g for 1 hour to obtain purified EGa1 cell membranes.

[0081] Preparation of LNP-MRTX1133 liposomes:

[0082] DPPC:DSPE-PEG5K:cholesterol was dissolved in chloroform at a molar ratio of 3:2:1. MRTX1133 was added, and the ratio of drug (MRTX1133) to liposomes was 0.5 mg:6 mg. The mixture was rotary evaporated to form a film, and then vacuum dried (lyophilized at -80°C for 2 hours, followed by vacuum freezing at -20°C for 10 hours) to remove the solvent. After resuspending in PBS, the film was sonicated to form LNP-MRTX1133.

[0083] Formation of hybrid nanovesicles:

[0084] 293T-EGa1 cell membrane solution was mixed with LNP-MRTX1133 at a mass ratio of 1:6, and extruded sequentially through 400 nm, 200 nm, and 100 nm polycarbonate membranes 15 times each to form EGa1-hNVs-M. Free drug was removed by centrifugation using a 100 kDa ultrafiltration tube to obtain purified EGa1-hNVs-M. In some other embodiments, 293T-EGa1 cell membrane solution was mixed with LNP-MRTX1133 at mass ratios of 1:3 and 1:12.

[0085] Characterization analysis:

[0086] The results are as follows Figure 2 As shown, the preparation process is as follows: Figure 2 As shown in a. Stable cell lines were established through lentiviral infection, and Western blot analysis was performed (…). Figure 2 (b) This verified the successful expression of the membrane-associated Myc tag EGa1. Furthermore, confocal microscopy results from immunofluorescence experiments showed that the Myc tag EGa1 was uniformly distributed on the surface of the lentivirus-infected 293T cell membrane. Figure 2c). Subsequently, cell membranes were extracted from 293T-EGa1 cells using gradient centrifugation and co-extruded with Liposome@MRTX1133 to form EGa1-hNVs-M. Notably, hybrid vesicles with different cell membrane-to-liposome ratios (1:3, 1:6, 1:12) were successfully prepared. Dynamic light scattering (DLS) analysis ( Figure 2 d) showed no significant difference in particle size among the three groups, with an average vesicle diameter of approximately 91.28 nm. However, as the proportion of cell membrane in the heterozygous vesicles increased, the zeta potential gradually decreased from -26.2 mV to -38.7 mV. Figure 2 e). These results were further confirmed by observations using transmission electron microscopy (TEM). Figure 2 f). Cell uptake kinetics were assessed by co-incubating A427 cells with hybrid nanovesicles at a lipid-to-membrane ratio of 1:3 and performing flow cytometry analysis at different time points. Compared with Liposomes, EGa1-hNVs exhibited significantly enhanced cell uptake efficiency, with a marked increase in intracellular fluorescence intensity observed after 12 hours of incubation. Figure 2 These results indicate that EGFR-targeting heterozygous vesicles possess superior time-dependent endocytosis in KRAS-mutant NSCLC cells. Based on quantitative uptake analysis, A427 cells were co-incubated with Cy5.5-loaded heterozygous liposomes for 24 hours, and cell internalization was observed using confocal fluorescence microscopy. Figure 2 h). Quantitative analysis of relative fluorescence intensity (RFI) was then performed. Figure 2 The results showed that the heterozygous vesicles with a membrane lipid ratio of 1:6 produced significantly higher fluorescence signals in the cells than other groups, further confirming their higher uptake efficiency. Based on these results, EGa1-hNVs with a membrane lipid ratio of 1:6 were selected for subsequent in vitro experiments. Furthermore, a standard curve for Western blot analysis was established using serial dilutions of the Myc-tagged fusion protein. Figure 2 (j). Subsequently, a series of different drug concentrations were co-incubated with a fixed amount (6 mg) of liposomes to evaluate their encapsulation efficiency (EE%) and drug loading (DL%). Among all test conditions, the formulation with a drug loading of 0.5 mg performed best, achieving an encapsulation efficiency of 77.17% and a drug loading of 62.26%, thus determining the optimal drug-to-liposome ratio for subsequent experiments. Figure 2 Further characterization of the EGa1-hNVs-M formulation showed that the system possesses good physicochemical stability and is suitable for in vivo experiments. Its particle size remained stable, and the cumulative drug release curves were similar under physiological conditions (pH 7.4) and acidic conditions (pH 5.6), demonstrating good pH stability. Figure 2 l).

[0087] Example 2

[0088] This embodiment provides the cytotoxicity and signaling pathway changes of EGa1-hNVs-M prepared in Example 1.

[0089] Cytotoxicity test:

[0090] MRTX1133 was used at different concentration gradients (0–20 μM) to evaluate its selective cytotoxicity. Cell viability assays showed that MRTX1133 was effective against KRAS cells. G12D Mutant cell lines exhibit potent and specific cytotoxic activity, while having minimal impact on non-mutant cells. Figure 3 a). To evaluate the therapeutic potential of EGa1-hNVs-M, a series of in vitro experiments were conducted in A427 non-small cell lung cancer (NSCLC) cells. The cells were divided into three groups: a negative control group (NC), a free MRTX1133 group, and an EGa1-hNVs-M group. All treatment groups used a fixed concentration of 17 μM MRTX1133 (corresponding to the IC50 of A427 cells). 50 To ensure consistent drug exposure, the absorbance of A427 cells was measured using the CCK-8 assay after 24 hours of treatment. The results showed that EGa1-hNVs-M significantly enhanced the inhibitory effect on tumor cell proliferation compared to free MRTX1133 (p < 0.0001), highlighting the enhanced cytotoxicity effect brought about by nanocarrier delivery. Figure 3 b).

[0091] Signal path analysis:

[0092] Figure 3 c illustrates the typical EGFR-RAS signaling pathway and its main downstream pathways, including the PI3K-AKT and RAF-MEK-ERK cascades. These pathways play a crucial role in regulating tumor cell proliferation, survival, and immune escape. To investigate the changes in major oncogenic signaling pathways after drug treatment, Western blot analysis was performed at 3, 6, and 12 hours post-treatment. Figure 3d). Free MRTX1133 significantly inhibited the phosphorylation of AKT and ERK, indicating that it simultaneously inhibited the PI3K / AKT and MAPK / ERK signaling pathways. However, unexpected upregulation of phosphorylated EGFR (pEGFR) was observed after MRTX1133 treatment. This phenomenon may originate from a feedback regulatory mechanism, namely, MRTX1133 inhibits ERRFI1, a negative regulator of EGFR, thereby relieving ERRFI1's inhibitory effect on EGFR, leading to a compensatory increase in EGFR phosphorylation. Notably, the EGa1-hNVs-M group not only maintained the inhibitory effect on pAKT and pERK but also effectively alleviated the increase in pEGFR, demonstrating its advantage in overcoming adaptive resistance mechanisms. Furthermore, elevated PD-L1 expression levels were detected in both MRTX1133 treatment groups, suggesting a possible immune escape response. This result further supports the rationale for its combined use with PD-L1 blockade therapy in subsequent studies.

[0093] Example 3

[0094] This embodiment provides an in vivo mouse targeting and biodistribution assessment of EGa1-hNVs-M, including that prepared in Example 1.

[0095] Model building and grouping:

[0096] By carrying KRAS G12D The lentiviral expression construct was transfected into M109 mouse lung cancer cells to establish KRAS. G12D High expression (KRAS) G12D A KRAS-OE mouse lung cancer model was obtained. G12D -OE M109 cells. One week after subcutaneous tumor formation in nude mice, DiD-labeled EGa1-hNVs with different membrane-to-liposome ratios were administered via tail vein injection.

[0097] Experimental results:

[0098] IVIS imaging was used to dynamically monitor fluorescence signals in vivo in order to assess their distribution in vivo. Figure 4 a). 24 hours after injection, the mice were sacrificed, and the tumors and major organs were removed for in vitro imaging ( Figure 4 (b) to compare tissue-specific enrichment. Analysis of DiD fluorescence intensity in tumors ( Figure 4 c), and perform statistical evaluation of the tumor and major organs ( Figure 4(d, 4e) The results showed that EGa1-hNVs with a membrane-to-liposome ratio of 1:6 achieved the highest tumor enrichment, while exhibiting the least distribution in non-target tissues such as the liver. This result was further confirmed by frozen sections of tumor tissue and fluorescence microscopy. Figure 4 f). Quantitative analysis of relative fluorescence intensity (RFI) further validated the excellent tumor-targeting ability of the 1:6 formulation. Figure 4 g). These results clearly demonstrate that a membrane-to-liposome ratio of 1:6 is the optimal ratio for enhancing tumor enrichment, providing a reasonable basis for subsequent therapeutic research.

[0099] Example 4

[0100] This embodiment provides a comparative antitumor experiment of mouse subcutaneous xenograft tumor models, including EGa1-hNVs-M prepared in Example 1.

[0101] Model building and grouping:

[0102] Using KRAS G12D -OE M109 cells were used to establish subcutaneous tumors in C57BL / 6 mice. Mice were divided into five groups (n=8 per group) according to different drug treatment conditions: control group (G1), liposome group (G2), MRTX1133 group (G3), EGa1-hNVs-M group (G4), and EGa1-hNVs-MP group (G5). Figure 5 As shown in a. Five mice were randomly selected from each group to evaluate the treatment effect and long-term survival rate. The remaining three mice underwent tumor resection immediately after 14 days of treatment for subsequent experiments.

[0103] Experimental results:

[0104] The results showed that, compared with other groups, EGa1-hNVs-MP treatment significantly inhibited tumor growth and prolonged survival. Figure 5 b, 5c). TUNEL staining was used to assess apoptosis in tumor tissues ( Figure 5 d), quantitative analysis showed that the level of apoptotic cells in the EGa1-hNVs-MP group was significantly increased ( Figure 5 e). Literature studies have shown that MRTX1133 treatment can enhance the infiltration of CD8⁺ T cells in the tumor microenvironment. Based on this, this invention further investigates whether EGa1-hNVs-MP treatment can further promote the infiltration of CD8⁺ T cells in subcutaneous tumors. Tumor sections were subjected to dual-label, three-channel immunofluorescence staining to detect CD3⁺ and CD8⁺ T cells (e). Figure 5 f), and quantitative analysis of relative fluorescence intensity (RFI) was performed ( Figure 5 g). Compared with MRTX1133 monotherapy, EGa1-hNVs-MP treatment significantly increased CD8⁺ T cell infiltration in tumor tissue. To further verify this result, another group of mice with subcutaneous tumors were treated with the same administration regimen, and tumor tissue was collected and analyzed by flow cytometry. Figure 5 Quantitative analysis of the intratumoral CD8⁺ T cell population showed a significant increase in the EGa1-hNVs-MP group compared to the control group (h). Figure 5 These data further confirm that the combination therapy significantly promoted the infiltration of CD8⁺ T cells in the tumor microenvironment, highlighting its powerful immunomodulatory effect.

[0105] Example 5

[0106] This embodiment provides a comparative evaluation of a mouse lung metastasis model.

[0107] Model building and grouping:

[0108] Following encouraging results obtained in the subcutaneous tumor model, this invention establishes a more clinically relevant lung metastasis model via intravenous injection of KRAS. G12D -OE M109 cells were introduced into C57BL / 6 mice. Four days after tumor cell inoculation, mice were randomly divided into five groups (n = 5 per group): control group (G1), liposome group (G2), MRTX1133 group (G3), EGa1-hNVs-M group (G4), and EGa1-hNVs-MP group (G5). Treatment in each group was administered via tail vein injection twice weekly for two weeks. Figure 6 a).

[0109] Experimental results:

[0110] like Figure 6 As shown in b, there were no significant differences in body weight changes among the groups of mice during treatment, indicating that all formulations were well tolerated. More notably, survival analysis ( Figure 6 c) The results showed that EGa1-hNVs-MP treatment significantly prolonged overall survival, highlighting the therapeutic advantages of the EGa1-hNVs-MP strategy. Lung tissue was collected for pathological evaluation after completion of the treatment regimen. Macroscopic observation ( Figure 6 d) and nodule count ( Figure 6 (e) showed that the lung metastatic burden was significantly reduced in the EGa1-hNVs-MP group compared to the other groups. Subsequent hematoxylin and eosin (H&E) staining further confirmed the reduction in tumor infiltration area in the lung tissue, consistent with macroscopic observations. Figure 6f, 6g). Based on histological findings, this invention performed immunohistochemical (IHC) analysis to detect PD-L1 (f, 6g) in different treatment groups. Figure 6 h, 6i) and phosphorylated ERK (pERK) Figure 6 The expression patterns of PD-L1 (j, 6k) were observed. In both the MRTX1133 monotherapy group and the EGFR-targeted therapy group, PD-L1 expression was significantly increased, suggesting that targeted inhibition of KRAS may trigger immune escape mechanisms in the tumor microenvironment. In contrast, EGa1-hNVs-MP treatment significantly inhibited PD-L1 expression, which may explain the enhanced CD8⁺ T cell infiltration in this group. Furthermore, this combined strategy showed more significant inhibition of pERK signaling, reflecting its more effective blocking effect on RAS pathway activation and tumor growth.

[0111] Example 6

[0112] This embodiment provides a spontaneous KRAS G12D Comparative validation of mutation-positive lung cancer models

[0113] Model building and grouping:

[0114] This invention evaluated the efficacy of EGa1-hNVs-MP therapy using a KRAS-driven orthotopic lung cancer transgenic mouse model. In this model, CC10-rtTA / TetO-Cre / LSL-Kras... G12D Mice were given doxycycline (DOX) in their drinking water starting at 6–8 weeks of age to induce tumor formation. After tumor establishment, mice were randomly assigned to receive PBS (G1), free MRTX1133 (G2), or EGa1-hNVs-MP (G3) for in vivo comparative analysis. Treatment was administered via tail vein injection twice weekly for four weeks. Figure 7 a).

[0115] Experimental results:

[0116] The results showed that, compared with other groups, EGa1-hNVs-MP treatment significantly prolonged overall survival. Figure 7 (b) and the weight did not fluctuate significantly, indicating that it has both good therapeutic effect and good tolerability. Figure 7 c). Twenty-eight days after the start of treatment, mouse lung tissue was collected, fixed overnight with Bouin reagent, and photographed to assess macroscopic tumor burden ( Figure 7 d). Statistical analysis of the number of lung surface tumor nodules showed that the EGa1-hNVs-MP group significantly reduced the number of visible lung nodules, with an average of approximately 4 nodules per mouse. Figure 7e). In addition, hematoxylin and eosin (H&E) staining was performed on paraffin-embedded lung tissue sections. Figure 7 f), and quantitatively analyzed the proportion of tumor area to total lung area. Compared with other groups, EGa1-hNVs-MP treatment significantly reduced the overall tumor burden (f). Figure 7 g). TUNEL staining ( Figure 7 h) further confirmed that the number of apoptotic cells in the tumor region of the EGa1-hNVs-MP group was significantly increased ( Figure 7 i), demonstrating its role in promoting tumor cell apoptosis. This supports the superior anti-tumor efficacy of this combined nanotherapy strategy. At the molecular level, the therapeutic effect was further validated by multiplex immunofluorescence staining, detecting markers including PD-L1, phosphorylated EGFR (pEGFR), phosphorylated ERK (pERK), and DAPI (for nuclear visualization). Figure 7 The results showed that although MRTX1133 monotherapy effectively inhibited downstream RAS signaling (manifested by decreased pERK levels), it still induced upregulation of PD-L1 and pEGFR expression. In contrast, EGa1-hNVs-MP treatment significantly downregulated these three markers, indicating that this strategy simultaneously blocked feedback activation and downstream signaling pathways. Figure 7 (k–m). This combined inhibition of a pair of key oncogenic and immunosuppressive signals highlights the therapeutic potential and translational value of this strategy in KRAS-mutant NSCLC.

[0117] As can be seen from Examples 1-5, the EGa1-hNVs-MP composite therapeutic agent of the present invention has the following technical advantages:

[0118] Precise targeting: EGa1 ligand-mediated membrane fusion improves the recognition and uptake efficiency of KRAS mutant cells;

[0119] High drug loading and stability: Nano-hybrid structures improve drug encapsulation efficiency and in vivo stability;

[0120] Synergistic anti-tumor effect: KRAS inhibition and PD-L1 immune blockade synergistically enhance the therapeutic effect;

[0121] Drug resistance reversal: Effectively inhibits EGFR feedback activation and improves the problem of resistance to MRTX1133 monotherapy;

[0122] Significantly prolongs survival: It exhibits superior anti-tumor effects in both xenograft tumors and spontaneous models.

[0123] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An EGFR-targeted nanovesicle co-system, characterized in that, Including nanovesicle units and immune checkpoint inhibitors; The nanovesicle unit includes hybrid nanovesicles, EGFR-specific nanobodies, and KRAS-specific inhibitors.

2. The EGFR-targeted nanovesicle combined system of claim 1, characterized in that, The hybrid nanovesicles are membrane-mimicking structures, formed by the co-assembly of synthetic lipid components and natural cell membrane-derived components. The EGFR-specific nanobody is attached to the surface of the hybrid nanovesicles via covalent or coupling bonds. The KRAS-specific inhibitor is encapsulated within the vesicles of the hybrid nanovesicles.

3. The EGFR-targeted nanovesicle combined system of claim 1, characterized in that, The KRAS-specific inhibitors include KRAS. G12D Specific inhibitors; The KRAS G12D Specific inhibitors include MRTX1133; The immune checkpoint inhibitors include anti-PD-L1 antibodies; The anti-PD-L1 antibody includes InVivoMAb anti-mouse PD-L1 antibody.

4. An EGFR-targeted nanovesicle combined system according to any one of claims 1-3, characterized in that, The preparation method of the nanovesicle unit includes the following steps: Preparation of cell membrane solutions expressing EGFR-specific nanobodies; Prepare liposome solutions containing KRAS-specific inhibitors; Cell membrane solution and liposome solution are mixed and extruded through a nanofiltration membrane to form nanovesicle units.

5. An EGFR-targeted nanovesicle combined system as described in claim 4, characterized in that, The preparation method of the nanovesicle unit further includes the following steps: After forming nanovesicle units, the free drug was removed by centrifugation through a 50-150 kDa ultrafiltration tube to obtain purified nanovesicle units.

6. A combined system for targeting EGFR nanovesicles as described in claim 4, characterized in that, The preparation of the cell membrane solution includes the following steps: Using a vector containing EGFR-specific nanobodies, tool cells were transfected with lentiviruses, and cell lines that stably expressed EGFR-specific nanobodies were screened to obtain cell lines. Cell lines that stably expressed EGFR-specific nanobodies were collected, suspended in Tris buffer, sonicated, centrifuged to remove organelle residues, and then centrifuged again to obtain purified cell membranes, thus obtaining cell membrane solutions expressing EGFR-specific nanobodies. The preparation of the liposome solution includes the following steps: Synthetic phospholipids, phospholipid-polymer conjugates, and cholesterol were dissolved, and a KRAS-specific inhibitor was added. The mixture was then rotary evaporated to form a film. After vacuum drying to remove the solvent, the film was resuspended in phosphate buffer solution and sonicated to form a liposome solution containing the KRAS-specific inhibitor.

7. An EGFR-targeted nanovesicle combined system as described in claim 6, characterized in that, The ratio of the cell membrane solution to the liposome solution is 1:(3~12) by mass. The nanofiltration membrane extrusion includes the following steps: extruding nanofiltration membranes of 400 nm, 200 nm and 100 nm sequentially 10 to 20 times respectively; The nanofiltration membrane includes a polycarbonate membrane.

8. An EGFR-targeted nanovesicle combined system as described in claim 6, characterized in that, The carrier containing EGFR-specific nanobodies includes the pCDH-CMV-EGa1-VHH-Myc-PDGFRβTM-Puro carrier. The tool cells include 293T cells; The process of selecting cell lines that stably express EGFR-specific nanobodies includes the following steps: Western blot and immunofluorescence detection showed that the Myc tag signal indicated that the EGFR-specific nanobody was successfully localized on the cell membrane surface, which is a cell line that stably expresses the EGFR-specific nanobody. The Tris buffer contains potassium chloride, magnesium chloride, Tris, and a protease inhibitor. The molar ratio of potassium chloride, magnesium chloride, and Tris in the Tris buffer solution is (3~7):1:(8~12). The volume ratio of the protease inhibitor to the cell membrane solution is (80~120):1; The ultrasonic fragmentation temperature is 2~6℃, and the time is 8~12 minutes; The centrifugation speed for removing organelle residues is 8000~12000×g, and the centrifugation time is 8~12 minutes; The centrifugation speed for obtaining the purified cell membrane after further centrifugation is 80,000~120,000×g, and the centrifugation time is 0.5~1h.

9. An EGFR-targeted nanovesicle combined system as described in claim 6, characterized in that, The synthetic phospholipids include DPPC; The phospholipid-polymer conjugate includes DSPE-PEG5K; The molar ratio of the synthetic phospholipid, phospholipid-polymer conjugate, and cholesterol is (1-5):(1-3):1; The solvents used to synthesize phospholipids, phospholipid-polymer conjugates, and cholesterol include chloroform; The mass ratio of the KRAS-specific inhibitor to the liposome is (0.5~6mg):6mg; The step of removing solvent by vacuum drying includes: Freeze-dry at -70~-90℃ for 2~5 hours; Vacuum freezing at -20~-25℃ for 6~10 hours.

10. The use of the EGFR-targeting nanovesicle combination system according to any one of claims 1-3 in the preparation of a combination therapy agent for KRAS-mutant non-small cell lung cancer.

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