A bifunctional nanomedicine based on autophagy-targeting chimeric T-cell connector, its preparation method and application

Abstract

This invention relates to the field of nanomedicine technology, specifically to a bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector, its preparation method, and its applications. The bifunctional nanomedicine comprises a lipid shell with an anti-CD3 antibody conjugated thereon; a T-cell receptor targeting mutant p53 protein inserted into the lipid shell; and the drug KPY encapsulated within the lipid shell, as well as PFP encapsulated therein. The nanomedicine specifically recognizes KRAS / TP53 mutant tumors via the surface-anchored T-cell receptor. Low-intensity focused ultrasound (HIFU) induces a PFP phase transition, leading to the release of KPY at the tumor site to induce intracellular KRAS degradation. The CD3 antibody activates CD8+. + T cells generate extracellular immune activation. This technical solution can solve the technical problem of the lack of targeted drugs for tumors associated with KRAS / TP53 co-mutations in existing technologies, and has ideal prospects for widespread application.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, specifically to a bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector, its preparation method, and its application. Background Technology

[0002] Gene mutations are one of the core biological mechanisms driving tumor development and treatment resistance. They not only reshape the malignant phenotype of tumor cells but also directly affect treatment response and patient prognosis. Co-mutations of the KRAS and TP53 genes are a particularly challenging molecular subtype in clinical practice. These co-mutated tumors often exhibit higher malignancy, such as stronger invasive and metastatic capabilities, faster proliferation rates, and poorer clinical prognosis, and have long lacked precise targeted therapies. With the rising global cancer incidence, the disease burden caused by KRAS / TP53 co-mutation-related tumors continues to increase, becoming a major challenge restricting breakthroughs in cancer treatment and threatening public health. Therefore, developing safe, efficient, and targeted treatment strategies for these co-mutated tumors has become a pressing issue in current cancer research.

[0003] Clinically, KRAS / TP53 co-mutations are widely present in various refractory tumors, most commonly pancreatic cancer (PDAC), colorectal cancer (CRC), and non-small cell lung cancer (NSCLC). These three types of cancer are inherently difficult to treat and have high recurrence rates, and the presence of co-mutations further exacerbates the treatment challenges. Although there are differences in treatment response—for example, some NSCLC patients with KRAS / TP53 co-mutations have relatively higher response rates to immune checkpoint inhibitors (ICIs)—the benefits of immunotherapy for these co-mutated patients in pancreatic and colorectal cancers remain very limited, and most patients fail to achieve long-term survival benefits.

[0004] In the field of targeted therapy, existing drugs also have significant shortcomings. Currently, the only approved KRAS-targeting drug, sotorasib (trade name Lumakras), only targets KRAS. G12C This specific mutation subtype is effective, precisely inhibiting the growth of tumors associated with that mutation; however, KRAS is more prevalent in clinical practice. G12D KRAS G12V Some KRAS mutation subtypes are completely insensitive to this drug, resulting in only a very small number of KRAS-mutant patients benefiting from it. As for TP53 mutations, due to their unique protein structure and complex function, drugs that directly target TP53 mutations are still in the early stages of research and development, and there is no clear clinical efficacy data to support their application potential.

[0005] More importantly, KRAS and TP53 mutations do not have a simple additive effect in tumors, but rather exhibit a significant synergistic oncogenic effect: KRAS mutations drive abnormal cell proliferation, while TP53 mutations impair their tumor-suppressive function, leading to inhibited apoptosis and defective DNA damage repair. Together, they enhance the malignant phenotype and drug resistance of tumors. This characteristic means that inhibitory therapy targeting only a single pathway (such as KRAS or TP53) is insufficient to break down tumor drug resistance mechanisms, let alone achieve sustained anti-tumor effects. Therefore, further exploration of novel therapeutic strategies for KRAS / TP53 co-mutated tumors is urgently needed. Summary of the Invention

[0006] The present invention aims to provide a bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector to address the technical problem of the lack of targeted drugs for tumors associated with KRAS / TP53 co-mutation in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector comprises a lipid shell; an anti-CD3 antibody is coupled to the lipid shell; and a T-cell receptor targeting mutant p53 protein is inserted into the lipid shell. The lipid shell encapsulates a drug KPY with the structural formula shown in Formula VII:

[0008] Formula VII The lipid shell also encapsulates PFP.

[0009] Furthermore, the T-cell receptor targeting mutant p53 protein includes a T-cell receptor protein with a gene sequence as shown in SEQ ID NO. 3 for targeting R175H mutant p53 protein, and a T-cell receptor protein with a gene sequence as shown in SEQ ID NO. 4 for targeting R248Q mutant p53 protein.

[0010] Furthermore, the drug KPY is formed by linking KRB peptide to YOK-1304 via PEG2000; The structure of the KRB polypeptide is: Ac-KRRRRCPLYISYDPVCRRRRK(Biotin)-NH2; Ac represents the N-terminal acetyl group; the C-terminal carboxyl group of the KRB polypeptide is acetylated; the two cysteine ​​C residues in the KRB polypeptide are linked by a disulfide bond; Biotin indicates that the side chain amino group of lysine K is modified with biotin.

[0011] Furthermore, the drug KPY is prepared by the following method: S1: Synthesis of YOK-NH2: YOK-1304 was reacted with BOC-6-aminohexanoic acid, EDC and DMAP in DMF solvent; after the reaction was completed, the solvent was removed and the reaction was carried out in DCM containing TFA; after the reaction was completed, YOK-NH2 was obtained by purification and drying. S2: Synthesis of YOK-PEG2000-COOH: YOK-NH2 reacts with NHS-PEG2000-COOH and TEA in solvent DCM; after the reaction is completed, YOK-PEG2000-COOH is obtained by purification and drying. S3: Synthesis of target drug KPY: YOK-PEG2000-COOH is reacted with EDC and NHS in DMF solvent; after the reaction is completed, the solvent is removed and the product is dissolved in DCM solvent; after washing with water, drying and solvent removal, it is reacted with KRB peptide and TEA in DMF solvent; after the reaction is completed, the drug KPY is obtained after purification and drying.

[0012] This technical solution also provides a method for preparing bifunctional nanomedicines based on autophagy-targeting chimeric T-cell connectors, characterized by comprising the following steps performed sequentially: SS1: Obtaining a cell membrane containing a chimeric T-cell receptor targeting the mutant p53 protein; SS2: SPC, Chol, DSPE-PEG2000, KPY, and PFP were dissolved in chloroform and formed into a membrane by rotary evaporation; after hydration treatment, DSPE-PEG-streptavidin was added to the membrane; after ultrasonic treatment and liposome extrusion treatment, drug-loaded avidin liposomes were obtained. SS3: Drug-loaded avidin liposomes were mixed with cell membranes, and after sonication and incubation, KPPF@TCR liposomes were obtained. SS4: KPPF@TCR liposomes and biotinylated anti-CD3 antibody were mixed and incubated to obtain a bifunctional nanomedicine based on autophagy-targeting chimeric T-cell connector.

[0013] Furthermore, the cell membrane of the T cell receptor chimeric with a target mutant p53 protein was extracted from TCR-T cells containing membrane proteins with co-expressed gene sequences as shown in SEQ ID NO. 3 and membrane proteins with gene sequences as shown in SEQ ID NO. 4.

[0014] Furthermore, the TCR-T cells are prepared by the following method: SSS1: CD8 + After T cells are activated by IL-2, the α and β chains of the cell's endogenous T cell receptor are knocked out, and CD8+ of the TCR is knocked out. + T cells; SSS2: The gene fragment with sequence such as SEQ ID NO. 3 and the gene fragment with sequence such as SEQ ID NO. 4 were integrated into the pWPXL expression vector to obtain the R175H-TCR-pWPXL expression vector and the R248Q-TCR-pWPXL expression vector, respectively. R175H-TCR lentivirus and R248Q-TCR lentivirus were constructed using the R175H-TCR-pWPXL expression vector and the R248Q-TCR-pWPXL expression vector, respectively. SSS3: TCR-T cells were obtained by co-infecting TCR-knockout CD8+ T cells with R175H-TCR lentivirus and R248Q-TCR lentivirus.

[0015] This technical solution also provides the application of a bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector in the preparation of drugs for treating KRAS / TP53 co-mutant tumors.

[0016] Furthermore, products for treating KRAS / TP53 comutated tumors include bifunctional nanomedicines based on autophagy-targeting chimeric-T-cell connectors, devices for delivering LIFU, and CD8... + T cells and IL-2.

[0017] Furthermore, in KRAS / TP53 co-mutated tumors, the KRAS mutation type includes KRAS... G12C Mutation, KRAS G12D Mutation, KRAS G12V Mutations; TP53 mutation types include TP53 R175H Mutation, TP53 R248Q Mutation, TP53 V128del Mutations; tumors with KRAS / TP53 co-mutations are pancreatic cancer, colorectal cancer, or non-small cell lung cancer.

[0018] In summary, the technical principle of this solution is as follows: This approach utilizes a bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector (AUTACE). Through T-cell receptors anchored on its surface, it specifically recognizes KRAS / TP53-mutant tumors. Low-intensity focused ultrasound (HIFU) induces a liquid-gas phase transition in PFP, enabling the spatiotemporal release of KPY at the tumor site to induce intracellular KRAS degradation. A CD3 antibody recruits and activates CD8. + T cells generate an "extracellular immune activation" effect; through the synergistic effect of "degradation + activation," precise and efficient treatment of KRAS / TP53 co-mutated tumors is achieved, as detailed below: (1) Overall technological improvement of nanomedicine In this technical solution, a T-cell receptor-T-cell connector (TCR-TCE) targeting the TP53 mutant antigen is used as a "guide" to deliver a KRAS-targeting protein degradation molecule that initiates autophagy to the tumor. Utilizing the synergistic effect of "intracellular KRAS degradation" and "extracellular immune activation," highly efficient killing of KRAS / TP53 co-mutated tumors is achieved. However, achieving efficient conjugation of the target protein degradation (TPD) with the T-cell connector still faces several challenges: First, traditional antibody-drug conjugates (ADCs) have limited loading capacity, making it difficult to achieve sufficient effective drug loading and tissue exposure; second, the T-cell connector, which combines target recognition and T-cell activation, can have its spatial conformation altered by drug loading, affecting its biological activity. This technical solution successfully solves these problems, specifically in the following two aspects: On the one hand, addressing the limited drug loading capacity of traditional ADCs, this approach innovatively employs liposomes as the core carrier. Utilizing the natural cavity structure of liposomes to construct a drug reservoir, it can efficiently encapsulate the KRAS-targeting TPD molecule KPY. Compared to ADCs, which typically load only 2-8 drug molecules, the liposome carrier in this approach allows for flexible adjustment of the KPY loading based on actual treatment needs, significantly increasing the effective drug content per unit carrier. This ensures that the drug reaches a therapeutic concentration sufficient to induce the degradation of the KRAS mutant protein after delivery to the tumor site, while avoiding problems such as insufficient tissue exposure and poor therapeutic effects due to insufficient drug loading. Furthermore, the excellent biocompatibility of the liposome carrier reduces non-specific adsorption and loss of the drug during delivery, further ensuring effective drug utilization.

[0019] On the other hand, addressing the challenge that drug loading may alter the spatial configuration of T-cell connectors and affect their biological activity, this approach breaks away from the traditional "direct drug-connector coupling" design concept and adopts a "functional module separation layout" strategy: the T-cell connectors responsible for target recognition and immune activation (including those targeting TP53) are integrated into the module layout. R175H TP53 R248Q Two mutated T-cell receptors (R175H-TCR and R248Q-TCR) and an anti-CD3 antibody were anchored to the surface of the liposome shell using biocompatible conjugation technology, maintaining their native spatial conformation and active sites. The TPD molecule KPY and the controlled-release substance PFP were co-encapsulated within the liposome cavity. This "surface functionalization-internal drug delivery" separation structure completely eliminates direct contact between KPY and the T-cell receptors and anti-CD3 antibody. This avoids spatial obstruction of the drug molecule's targeting recognition and activation sites on the T-cell connector, and prevents conformational changes caused by non-specific interactions between the drug and the T-cell receptors and anti-CD3 antibody. This ensures that the T-cell receptor targeting the mutated p53 protein can accurately recognize tumor cell surface antigens, and that the anti-CD3 antibody can efficiently recruit and activate CD8+.+ T cells, and KPY can be released on demand under LIFU triggering, achieving synergistic and efficient performance of the three major functions of targeting, activation and degradation.

[0020] (2) Innovative design of T-cell connectors Furthermore, this approach optimizes the design of the T-cell connector compared to existing technologies. T-cell receptor-based T-cell connector drugs (TCR-TCE) are novel immunotherapeutic molecules that combine the antigen recognition properties of T-cell receptors with immune activation mechanisms. In existing technologies, the core design of TCR-TCE involves fusing the antigen recognition domain of the T-cell receptor with the T-cell activation domain (such as an anti-CD3 antibody fragment) via a flexible linker to form a bifunctional molecule. In this technology, the T-cell receptor is anchored to a lipid shell by fusing the cell membrane containing the T-cell receptor with other lipids; the anti-CD3 antibody is coupled to the cell membrane via biotin-streptavidin binding, and the lipid-linked streptavidin is also interwoven within the lipid shell. Notably, the T-cell receptor and anti-CD3 antibody are not fused via a flexible linker, thus avoiding conformational abnormalities caused by fusion protein formation.

[0021] (3) Improvement of drugs targeting protein degradation This technical solution is the first to develop a drug, KPY, targeting the degradation of KRAS mutant proteins, and the first to achieve conjugation between the KRpep-2d peptide and YOK-1304. This design is a pioneering exploration in the field. During the research and development process, the team discovered a key technical challenge: YOK-1304 significantly interferes with the function of the KRB peptide obtained by optimizing the KRpep-2d peptide. If an inappropriate conjugation method is used, YOK-1304 will cause the KRB peptide to completely lose its binding ability to the KRAS mutant protein, resulting in the inability to effectively target and degrade the mutant KRB. This problem has become the core bottleneck restricting the drug's activity. To overcome this problem, the inventors tried various conjugation strategies between the KRB peptide and YOK-1304, and finally discovered that using PEG2000 as the conjugation bridging molecule can effectively preserve the nanomolar-level high affinity between the KRB peptide and the KRAS mutant protein, overcoming the negative impact of YOK-1304 on the binding activity of the KRB peptide. This discovery not only solves the technical challenge of coupling interference, but also ensures the highly efficient degradation activity of the targeted degradation drug against the target protein.

[0022] Compared with existing tumor treatment technologies, especially for the treatment of KRAS / TP53 co-mutated tumors, this invention has the following significant beneficial effects: (1) Address the technical deficiency of narrow target coverage and improve tumor adaptability. In existing technologies, KRAS-targeting drugs (such as sotorasib) only target KRAS. G12C Subtype-specific TP53-targeting drugs are still in the exploratory stage and cannot simultaneously adapt to multiple mutant subtypes. The nanomedicine of this invention can simultaneously target TP53. R175H and TP53 R248Q Mutations, and the ability to target KRAS, etc. G12C KRAS G12D KRAS G12V This nanoparticle targets and binds to various KRAS mutant proteins, including those with mutations, and degrades the target proteins. Its targets can cover mainstream KRAS / TP53 co-mutated tumors such as pancreatic cancer, colorectal cancer, and non-small cell lung cancer, significantly improving the drug's adaptability to different mutation subtypes and cancer types.

[0023] (2) Improve drug loading capacity and avoid functional interference, thereby ensuring therapeutic efficacy. This invention employs a separation design of surface-anchored functional molecules and internally encapsulated drugs: on the one hand, the cavity structure of the liposomes can significantly increase the drug loading of KPY, ensuring that the effective therapeutic concentration is achieved locally in the tumor; on the other hand, the T-cell receptor targeting the mutant p53 protein and the anti-CD3 antibody are anchored on the surface of the liposomes without spatial interference with the internally encapsulated KPY, thus completely preserving the targeting recognition ability of the T-cell receptor targeting the mutant p53 protein and the T-cell recruitment activity of the anti-CD3 antibody.

[0024] (3) Achieve spatiotemporally controllable release, taking into account both efficacy and safety. Existing chemotherapy or targeted drugs are prone to causing toxicity to normal tissues due to systemic distribution, and the drug release time cannot be precisely controlled. This invention uses a controlled release mechanism that triggers the PFP phase transition through LIFU to release KPY only at the tumor site. This not only increases the local drug concentration in the tumor to enhance efficacy, but also reduces the exposure of the drug in normal tissues, thereby reducing systemic toxicity and solving the clinical problem of balancing efficacy and safety.

[0025] (4) Construct a “dual-arm synergistic treatment” system KRAS and TP53 co-mutations synergistically enhance tumor drug resistance, and single-pathway inhibition is insufficient to achieve durable therapeutic effects. This invention utilizes KPY to degrade the KRAS mutant protein, targets the T-cell receptor of the mutant p53 protein to specifically recognize tumors, and activates CD8 with an anti-CD3 antibody. + T cells form a synergistic effect of "targeted degradation + activation of immune killing", which can act on both the internal driving pathways of tumor cells and the external immune microenvironment, breaking the drug resistance mechanism of comutated tumors and achieving a more durable anti-tumor effect.

[0026] In summary, this invention effectively solves multiple technical problems in existing technologies, such as the lack of specific drugs for KRAS / TP53 co-mutated tumors, narrow target coverage, low drug loading efficiency, high toxicity, and easy drug resistance, through structural, mechanistic, and application innovations. It provides a safe and efficient new strategy for the clinical treatment of these refractory tumors. Attached Figure Description

[0027] Figure 1 The results are obtained by liquid chromatography detection of the KRB peptide in Example 1.

[0028] Figure 2 The mass spectrometry results are for the KRB peptide in Example 1.

[0029] Figure 3 The mass spectrometry results are for KPY in Example 1.

[0030] Figure 4 The results are H-NMR detection results of KPY in Example 1.

[0031] Figure 5 The results of the affinity study between drugs such as KPY and KRAS series proteins in Example 2 are shown (a: KPY and KRAS). G12D Schematic diagram of protein-protein interaction; be: circular dichroism chromatogram results after KPY binds to KRAS protein; fi: affinity results between KPY and KRAS protein.

[0032] Figure 6 The results of the study on the effects of KPY on different tumor cell lines in Example 3 are as follows: (ad: Protein levels of KRAS and LC3 were detected by immunoblotting after cells were treated with different concentrations of KPY for 24 h; e: Distribution of KRAS and autophagosomes was detected by immunofluorescence after cells were treated with 20 μM KPY for 12 h; Data are expressed as mean ± standard deviation, n=20; *** indicates that compared with the corresponding blank group without KPY, p < 0.0001; fg: Protein levels of KRAS and LC3 were detected after cells were treated with KPY combined with HCQ for 24 h; h: KRAS protein level was detected after cells were treated with KPY combined with MG-132 for 24 h).

[0033] Figure 7 The results of flow cytometry analysis of the expression efficiency of R175H-TCR and R248Q-TCR proteins in TCR-T cells in Example 4 are shown.

[0034] Figure 8 This is a schematic diagram illustrating the preparation process of the bifunctional nanomedicine based on the autophagy-targeting chimeric T-cell connector in Example 5.

[0035] Figure 9Characterization of the bifunctional nanomedicine based on autophagy-targeting chimeric T-cell connector in Example 5 (a: transmission electron microscopy image; b: potential map; c: particle size distribution map; d: particle concentration; e: anti-CD3 antibody statistical graph; f: KPY release curve).

[0036] Figure 10 The experimental results of treating tumor cells with a bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector in Example 6 are shown in Figure 6 (a: cell immunofluorescence image; b: flow cytometry results; c, d: WB experimental results).

[0037] Figure 11 The experimental results of Example 6 are as follows: treatment of co-cultured tumor cells and T cells with bifunctional nanomedicine based on autophagy-targeting chimeric-T cell connector (a, b: statistical results of tumor cell killing rate treated with different nanoparticles under different effector-target ratios; cf: detection results of expression levels of CD69, IFN-γ, and GZMB; g: cell imaging results; data are expressed as mean ± standard deviation, n=3; *** indicates: compared with the corresponding blank group without KPY, p<0.0001).

[0038] Figure 12 The results of the tumor targeting experiment of the bifunctional nanomedicine based on autophagy-targeting chimeric-T cell connector in Example 7 are as follows (ad: in vivo imaging results; eh: in vitro imaging results of tumor sites; ik: in vitro imaging results of non-tumor sites; l: fluorescence imaging results of tumor sections; data are expressed as mean ± standard deviation, n=3; ** indicates: compared with the corresponding blank group without KPY, p<0.001).

[0039] Figure 13 The results of the in vivo antitumor effect experiment of the bifunctional nanomedicine based on autophagy-targeting chimeric-T cell connector in Example 8 are as follows (ad: experimental results for the PANC-1 subcutaneous xenograft tumor model; ef: experimental results for the MIA PaCa-2 subcutaneous xenograft tumor model).

[0040] Figure 14 The results of the biosafety evaluation of the bifunctional nanomedicine based on autophagy-targeting chimeric T-cell connector in Example 8 are shown in (a: serological markers; b: histological examination results). Detailed Implementation

[0041] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0042] To facilitate understanding of this technical solution, the relevant technical terms and concepts are explained in detail below: KRAS / TP53 comutated tumors refer to a type of tumor in which both KRAS and TP53 gene mutations occur simultaneously in the tumor cells. This comutation can significantly affect the malignancy, treatment response, and prognosis of the tumor.

[0043] The KRAS gene (Kirsten rat sarcoma viral oncogene homolog, GeneID: 3845) is a proto-oncogene that normally regulates cell growth, proliferation, and differentiation. Activating mutations can lead to uncontrolled cell proliferation, directly driving tumor development.

[0044] The TP53 gene (Tumor Protein P53) is a tumor suppressor gene that monitors DNA damage, prevents abnormal cell division, and induces apoptosis in damaged cells. If an inactivating mutation occurs, DNA damage accumulates, accelerating tumor development and making tumors more susceptible to drug resistance.

[0045] When KRAS and TP53 are mutated simultaneously, their effects are not simply additive, but rather produce a stronger malignant synergistic effect: enhancing tumor invasion and metastasis, reducing treatment sensitivity, and resulting in a worse prognosis.

[0046] KRAS / TP53 co-mutations are not limited to any particular type of tumor and have a high incidence in many solid tumors, most commonly including pancreatic cancer, colorectal cancer, non-small cell lung cancer, cholangiocarcinoma, and ovarian cancer. G12C Mutation: The glycine (G) at position 12 of the KRAS protein is mutated to cysteine ​​(C). The resulting protein is referred to as KRAS in the following description. G12C Protein. KRAS G12D Mutation: The glycine (G) at position 12 of the KRAS protein is mutated to aspartic acid (D). The resulting protein is referred to as KRAS in the following description. G12D Protein. KRAS G12V Mutation: The glycine (G) at position 12 of the KRAS protein is mutated to valine (V). The resulting protein is referred to as KRAS in the following description. G12VProteins. The G12C, G12D, and G12V mutations in the KRAS gene are all activating mutations at codon 12, representing the most common hotspot mutation types in the KRAS gene. The core of these mutations is an alteration of the amino acid at position 12 of the KRAS protein, leading to abnormal protein activation. The specific amino acid changes and structural differences directly affect the efficacy of targeted drugs. TP53 R175H Mutation: The mutation occurs in exon 5 of the TP53 gene, resulting in the replacement of arginine (R) with histidine (H) at amino acid position 175 of the p53 protein. TP53 R248Q Mutation: The mutation occurs in the 7th member of the TP53 gene, resulting in the replacement of arginine (R) with glutamine (Q) at amino acid position 248 of the p53 protein. This is one of the hotspot mutations in TP53. V128del Mutation: A frameshift mutation resulting in the deletion of valine (V) at amino acid position 128 of the p53 protein, causing premature termination of p53 protein translation and forming a truncated protein. TP53 R175H Mutation, TP53 R248Q Mutation, TP53 V128del These three mutations all lead to the loss of p53's tumor-suppressing function through different mechanisms. Among them, R175H and R248Q are missense mutations (combining loss and gain of function), while V128del is a frameshift mutation (complete loss of function). They indicate poor prognosis in various cancers and are closely related to treatment resistance, making them important targets for precision oncology diagnosis and targeted drug development.

[0047] The LC3-II / LC3-I ratio is a core molecular marker for assessing autophagy activity, used to quantitatively reflect the initiation and progression of the autophagy process. LC3 (Microtubule-associated protein 1A / 1B-light chain 3) is a key protein in autophagy, existing in two isoforms: LC3-I: the soluble, unmodified form in the cytoplasm; and LC3-II: the membrane-bound form of LC3-I after lipidation, which specifically localizes to the autophagosome membrane. During autophagy activation, LC3-I is modified into LC3-II by a series of enzymes and integrated into the autophagosome membrane. Therefore, the level of LC3-II directly reflects the number of autophagosomes formed. Changes in the LC3-II / LC3-I ratio can visually reflect the degree of autophagy activation: an increased ratio indicates activated autophagy and enhanced conversion of LC3-I to LC3-II; a decreased ratio indicates inhibited autophagy and reduced production or accumulation of LC3-II.

[0048] LIFU, short for Low-Intensity Focused Ultrasound, is a physical technology that utilizes focused ultrasound waves. Due to its low intensity, it is primarily used in the biomedical field to assist in drug delivery (such as promoting the penetration and release of nanoparticles into tumor tissue), regulate the tumor microenvironment, and enhance tissue permeability.

[0049] CD8 + T cells are an important subset of T lymphocytes, named for the CD8 molecule they express on their surface. Their core function is cytotoxicity: after recognizing cells infected by pathogens or tumor cells, they kill target cells by releasing substances such as perforin and granzymes, thus exerting anti-tumor and antiviral cellular immunity.

[0050] CD69 is an early activation marker of immune cells and belongs to the cell surface glycoprotein family. When immune cells such as T cells, B cells, and NK cells are activated, they rapidly express CD69, and therefore it is often used as a molecular indicator to determine whether immune cells are in an activated state.

[0051] CD3 is a key signaling complex on the surface of T cells, composed of CD3ε, γ, δ, and ζ subunits, which form a complex with the T cell receptor (TCR). When a T cell recognizes an antigen, CD3 is responsible for transmitting activation signals into the cell, serving as the signaling hub for T cell activation; anti-CD3 antibodies can regulate T cell function by binding to this molecule.

[0052] IFN-γ, or interferon-gamma, is an important cytokine. It is mainly produced by activated Th1 cells and CD8+ cells. + Secreted by T cells and NK cells, it has multiple functions.

[0053] GZMB, or Granzyme B, is a serine protease. It is primarily stored in CD8. + Granulase B is released into the cytotoxic particles of T cells and NK cells when these cells bind to target cells (such as tumor cells or infected cells), inducing apoptosis. It is a key killing molecule in cytotoxic immune responses.

[0054] IL-2: Interleukin-2 is a core cytokine in the immune regulatory network. It is mainly composed of activated CD4+. + Secreted by T cells, it is a growth factor for T cells and promotes CD8. + T cells, CD4 + The proliferation and activation of T cells enhance their cytotoxicity (such as secreting granzymes and perforin to kill target cells); at the same time, it maintains the survival and differentiation of T cells.

[0055] Example 1: Compound Synthesis (1) Synthesis of KRB KRpep-2d is an inhibitory cyclic peptide of KRAS that can bind to KRAS G12D, G12V, and G12C. Its CAS number is 2098181-84-9, and its sequence is: Ac-RRRRCPLYISYDPVCRRRR-NH2 (a disulfide bond is formed between Cys at positions 5 and 15, SEQ ID NO. 1). Its structural formula is shown in Formula I. Based on the macrocyclic peptide KRpep-2d that recognizes KRAS mutations, lysine residues are introduced at the N / C ends of this peptide, and the C-terminal lysine residue is coupled with biotin to obtain the KRB peptide, whose structural formula is shown in Formula I. Its specific sequence is as follows: Ac-KRRRR-cyclo(CPLYISYDPVC)-RRRRK(Biotin)-NH2 (KRB polypeptide, SEQ ID NO. 2); N-terminus: AC stands for acetyl (-COCH3), which modifies the N-terminus (free amino group) of the peptide. Its function is to enhance the stability of the peptide (avoid degradation by aminopeptidase) and reduce non-specific binding, which is a conventional modification method in the prior art. C-terminus: NH2 indicates that the C-terminal carboxyl group (-COOH) is amidated and converted into an amide group (-CONH2), which is also used to improve stability (resist degradation by carboxypeptidase) and to mimic the terminal structure of the natural peptide, which is a conventional modification method in the prior art. "cyclo()" indicates that the sequence in parentheses forms a cyclic structure, and a disulfide bond (-SS-) is formed to close the ring through the thiol group (-SH) of the cysteine ​​(C) at both ends. Based on SEQ ID NO.1, a lysine K is introduced at both the N-terminus and C-terminus to form the amino acid sequence of SEQ ID NO. 2. K (Biotin) means that the side chain amino group of the lysine (K) at the C-terminus is linked to a biotin tag, which facilitates subsequent detection.

[0056] KRB peptides can be synthesized using conventional peptide synthesis methods, and can be commissioned to a biotechnology company to synthesize them according to the above sequence. Details of the liquid chromatography detection results of the obtained KRB peptides can be found in [link to relevant documentation]. Figure 1 For detailed mass spectrometry results, please refer to [link / reference]. Figure 2 .

[0057]

[0058] Formula I (KRpep-2d polypeptide)

[0059] Formula II (KRB polypeptide) (2) Synthesis of KPY This step involves linking YOK-1304 to the KRB peptide. YOK-1304 has the CAS number 2409960-03-6 and its structural formula is shown in Formula III. YOK-1304 is a ligand for the p62-ZZ domain. YOK-1304 can activate p62-dependent selective macroautophagy.

[0060]

[0061] Formula III (YOK-1304) 10 mg of YOK-1304 was weighed and dissolved in 1 ml of N,N-dimethylformamide (DMF). Tert-butyloxycarbonyl-6-aminohexanoic acid (BOC-6-aminohexanoic acid, BOC being the protecting group for the amino group, 1.1 eq.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 2.0 eq.), and 4-dimethylaminopyridine (DMAP, 1.5 eq.) were added and dissolved completely. The reaction was carried out at room temperature for 12 h. The solvent was removed by vacuum distillation, and 2 ml of dichloromethane (CH2Cl2, DCM) was added for dispersion. 0.5 ml of trifluoroacetic acid (TFA) was added, and the reaction was carried out at room temperature for 0.5 h. The mixture was washed three times with water, dried over anhydrous sodium sulfate, and the reaction solution was transferred to a large amount of ice-cold diethyl ether to precipitate. The product was collected by filtration and dried under vacuum to obtain YOK-NH2 (see Formula IV for the reaction process).

[0062]

[0063] Formula IV Weigh 5 mg of YOK-NH2 and dissolve it in 1 ml of CH2Cl2. Add succinimide succinate-polyethylene glycol-carboxyl group (NHS-PEG2000-COOH, 1.1 eq.) and triethylamine (TEA, 2.0 eq.) and react at room temperature for 4 h. Wash three times with water, dry with anhydrous sodium sulfate, transfer the reaction solution to a large amount of ice-cold diethyl ether to precipitate, filter and collect the product, and dry under vacuum to obtain the YOK-PEG2000-COOH product (see Formula V for the reaction process).

[0064]

[0065] Formula V Weigh 5 mg of YOK-PEG2000-COOH and dissolve it in 1 ml of N,N-dimethylformamide (DMF). Add EDC (2.0 eq.) and N-hydroxysuccinimide (NHS, 2.0 eq.) and dissolve completely. React at room temperature for 12 h. Remove the solvent by vacuum distillation. Add 1 ml of CH2Cl2 to dissolve the product. Wash three times with water. Dry with anhydrous sodium sulfate and remove the solvent by vacuum distillation. Redissolve the product with 1 ml of DMF. Add KRB peptide (1.1 eq.) and triethylamine (3.0 eq.) and dissolve completely. React at room temperature for 12 h. Transfer the reaction solution to a dialysis bag (molecular weight cutoff 3500 Da) and dialyze in pure water for 12 h. Freeze-dry and solidify with ice-cold ether. Vacuum dry to obtain KPY (see Formula VI for reaction process details). The structural formula of KPY is shown in Formula VII. It is a compound formed by linking the amide group of the N-terminal lysine of the KRB peptide to YOK-1304 via PEG2000. Mass spectrometry (MS / MS) was used to analyze the KPY composition. Figure 3 ) and H-NMR detection of product size and structure ( Figure 4 ).

[0066]

[0067] Formula VI

[0068] Formula VII Example 2: Detection of the binding of KPY to mutant KRAS protein With KRAS G12D The protein was used as the receptor, and KPY as the ligand. AlphaFold3 was used to predict the receptor structure and select the best model. AutoDock Vina was used to prepare the receptor (ligand / water removal, residue addition, hydrogenation, Gasteiger charging, PDBQT) and ligand (3D model construction, hydrogenation, physiological pH ionization, energy minimization, PDBQT). After docking, the model was screened based on binding energy and key interactions, and visualized using PyMOL. Molecular docking simulations showed that KPY mainly interacts with KRAS. G12D The hydrogen bonds and hydrophobic interactions of the Q61, H95, Y96, Q99, R73, and V103 residues achieve binding. Figure 5 a).

[0069] KRAS G12D Protein (0.42 mmol, 1 mL), KRAS G12C Protein (0.42 mmol, 1 mL), KRAS G12VProtein (0.45 mmol, 1 mL) and KRAS protein (0.45 mmol, 1 mL) were incubated with KPY (5 mg / mL, 20 μL), KRB peptide (1 mg / mL, 100 μL), or YOK-1304 (10 mM, 23 μL) at 37 °C for 30 min, respectively. Protein absorbance was measured using a Jasco spectrophotometer. Results showed that KPY induced changes in the secondary structure of mutant KRAS (three mutations) upon binding; however, it did not bind to wild-type KRAS. Figure 5 be).

[0070] Biotinylated KRAS mutant protein (10 μg / mL) was loaded into a conventional streptavidin (SA) sensor (target 1.5 nm). The sensor was pre-washed in kinetic buffer (phosphate-buffered saline PBS + 0.1% Tween-20 + 5% bovine serum albumin BSA) for 120 s to establish a baseline. KPY was detected at a 2-fold gradient (e.g., 100–6.25 nM), recording association 120 s / dissociation 240 s. Background was removed using the reference channel, and a 1:1 model was fitted to KPY. D (Dissociation constant). Affinity results show that KPY is compatible with KRAS. G12D Protein, KRAS G12V Protein, KRAS G12C The binding affinity of the proteins is all in the nM range ( Figure 5 (fi), but KPY does not have an affinity for wild-type KRAS.

[0071] This technical solution is the first to construct a drug molecule conjugated with KRpep-2d peptide and YOK-1304. To ensure that the obtained composite molecule has the ideal binding effect on KRAS mutant protein, the inventors added lysine residues to the N-terminus and C-terminus of the KRpep-2d peptide to form the KRB peptide. The amide group of the lysine residue at the N-terminus of the KRB peptide is used for conjugation with YOK-1304. However, it was found in the study that the conjugation method of the KRB peptide and YOK-1304 seriously affected the affinity of the KRB peptide for the KRAS mutant protein. Inappropriate conjugation method will cause the KRB peptide to lose its ability to bind to the KRAS mutant protein. The inventors tried to conjugate the KRB peptide and YOK-1304 with PEG4, PEG8, PEG1000 and PEG2000. In actual operation, the method of Example 1 is followed, except that NHS-PEG4COOH, NHS-PEG8COOH, NHS-PEG100-COOH and NHS-PEG2000-COOH are used for the corresponding steps. The obtained products were KRB-PEG4-YOK-1304, KRB-PEG8-YOK-1304, KRB-PEG1000-YOK-1304, and KRB (i.e., the compound represented by formula VII). Affinity results showed that KRB-PEG4-YOK-1304 and KRB-PEG8-YOK-1304 did not specifically bind to mutant KRAS; while KRB-PEG1000-YOK-1304 had an affinity for mutant KRAS in the μM range (Table 1). Overall, PEG4 and PEG8 significantly weakened the binding effect, and no specific binding was observed within the test range (KRB). D =NS); PEG1000 still causes affinity to drop to the μM level; only PEG2000 can effectively retain nM level affinity.

[0072] Table 1: Affinity (K) of different compounds D Test results

[0073] Example 3: Degradation of mutant KRAS by KPY-induced autophagy Tumor cells were treated with different concentrations of KPY (0-20 μM) for 24 hours. The tumor cells included: PNAC-1 cells: carrying KRAS G12D and TP53 R175H Double mutation; belongs to human pancreatic cancer cell line, used for KRAS research. G12D With TP53 R175H The mechanism of pancreatic cancer development driven by dual mutations is an important model for exploring targeted therapy for this subtype of pancreatic cancer.

[0074] MIA PaCa-2 cells: carrying KRAS G12C and TP53 R248Q Double mutation; belonging to a human pancreatic cancer cell line, it is a classic model for pancreatic cancer research, often used to analyze tumor progression, metastatic potential, and radiotherapy and chemotherapy resistance mechanisms in pancreatic ductal adenocarcinoma, and can also be used to target KRAS. G12C Or TP53 R248Q Drug efficacy evaluation.

[0075] SK-CO-1 cells: carrying KRAS G12V The mutation, TP53, is wild-type (WT); it belongs to a human colorectal adenocarcinoma cell line and is used for research on the invasion and metastasis mechanisms of colorectal cancer, as well as KRAS. G12V The efficacy screening of targeted therapies can also be used to explore the association between the colorectal cancer microenvironment and tumor progression.

[0076] HCC827 cells: carrying TP53 V128del The mutation, KRAS is wild-type; it belongs to human non-small cell lung cancer cells and is the core model for studying targeted therapy and drug resistance mechanisms of non-small cell lung cancer. It is also used to explore basic research such as the lung cancer microenvironment and metastatic potential.

[0077] After treating tumor cells with different concentrations of KPY (0-20 μM) for 24 h, mutant KRAS expression decreased, while wild-type KRAS remained unaffected. Simultaneously, the LC3-II / LC3-I ratio (reflecting autophagy activity) increased, and KRAS co-localized with autophagosomes. Figure 6 ae). The addition of the autophagy inhibitor HCQ (hydroxychloroquine) reversed the downregulation of KRAS, while the proteasome inhibitor MG-132 had no such effect. Figure 6 (fh). This indicates that KPY degrades mutant KRAS via the autophagy-lysosomal pathway.

[0078] Example 4: Preparation of TCR-T cells and acquisition of cell membranes This technical solution constructs bispecific TCR-T cells that target both the R175H mutant p53 protein and the R248Q mutant p53 protein. The specific process is as follows: (1) CD8 + T cell (cytotoxic T lymphocyte) isolation Add 15 ml of lymphocyte separation medium (Stemcell, catalog number 18061) to a lymphocyte separation tube (Stemcell, catalog number 85460) and centrifuge at 350 g for 1 min. Take a 50 ml sterile centrifuge tube and add blood and PBS-Plus (PBS + 10% FBS + 0.1 mM EDTA). Dilute the blood and PBS-Plus at a 1:1 volume ratio and mix by inverting 2-3 times. Slowly add the diluted blood along the wall of the centrifuge tube to the lymphocyte separation tube mentioned above, and centrifuge at 1200 g for 10 min at 25°C. Take a new 50 ml sterile centrifuge tube, add the supernatant obtained from the previous centrifugation, and then add PBS-Plus to 50 ml. Centrifuge at 300 g for 15 min. Retain 5 ml of liquid, discard the rest of the supernatant, resuspend the cells, transfer to a 15 ml sterile centrifuge tube, and centrifuge at 300 g for 10 min. Discard the supernatant, resuspend the cells in 10 ml of PBS, and centrifuge at 300 g for 10 min. Discard the supernatant again, resuspend the cells in 1 ml of PBS to obtain PBMCs (peripheral blood mononuclear cells). Take a certain amount of PBMCs and analyze them using EasySep Human CD8. + According to the instructions, the T Cell Enrichment Kit (Stemcell, item number 19053) was used to sort and obtain CD8. + T cells.

[0079] (2) Knock out CD8 + T cell intrinsic T cell receptor (TCR) α and β chains Cell activation: 2×10 6 CD8 + Add 10 μl of T Cell TransAct (Miltenyi, catalog number 130-111-160) to TMB medium containing 300 U / ml IL-2 and activate for 48 h. TMB medium was prepared using RPMI 1640 as the basal medium, which contained: 10% fetal bovine serum (FBS), 1×GlutaMax (L-alanyl-L-glutamine, Gibco, catalog number 35050061), 1×MEM NEAA (non-essential amino acids, Gibco, catalog number 11140050), 25mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, Beyotime, catalog number C0217), 1×Penicillin-Streptomycin (penicillin-streptomycin bispecific antibody, Gibco, catalog number 15140122), 55µM β-mercaptoethanol, and 1× Sodium Pyruvate (sodium pyruvate, Gibco, catalog number 11360070).

[0080] Prepare the electroporation buffer: Add 0.64 µl (40 pmol) Cas9 protein, 1.8 µl (120 pmol) sgTRAC, 1.8 µl (120 pmol) sgTRBC, and 5 µl P3 Primary Cell 4D-Nucleofecto. TM X Kit, mix well and incubate at room temperature for 15 min to obtain electroporation solution.

[0081] Both sgTRAC and sgTRBC are sgRNAs (single guide RNAs). The former targets the TRAC gene (the constant region of the α chain of the T cell receptor) and is responsible for guiding Cas9 to a specific sequence of the TRAC gene; the latter targets the TRBC gene (the constant region of the β chain of the T cell receptor) and is responsible for guiding Cas9 to a specific sequence of the TRBC gene. The Cas9 protein and sgRNA form a stable Cas9-sgRNA complex (RNP complex). The sgRNA consists of two parts: a guide sequence and a scaffold sequence. In the complex, the scaffold sequence of the sgRNA binds to Cas9, while the guide sequence is exposed, allowing it to specifically recognize the target sequences of the intracellular TRAC and TRBC genes, ensuring precise cleavage.

[0082] The target sequence of sgTRAC for the TRAC gene is (corresponding to the guide sequence of sgTRAC): 5'-AGAGTCTCTCAGCTGGTACA-3' (SEQ ID NO. 1).

[0083] The target sequence of sgTRBC for the TRBC gene is (corresponding to the sgTRBC guide sequence): 5'-GGAGAATGACGAGTGGACCC-3' (SEQ ID NO. 2).

[0084] Once the target sequence of the sgRNA is determined, the corresponding sgRNA can be obtained using conventional methods available in existing technologies. In this technical solution, both sgTRAC and sgTRBC were synthesized by GenScript.

[0085] Take 1×10 6 An activated CD8 + T cells were added to the aforementioned electroporation solution and supplemented with P3 Primary Cell 4D-Nucleofector. TM Add the X kit to 20 µl; add the cell suspension to an electroporation cuvette and electroporate (T cell EH-115 program). After completion, culture the cells in TMB medium with 300 U / ml IL-2. After 5 days, flow cytometry was used to detect the knockout efficiency, and TCRKO-CD8 cells were obtained.+ T cells (TCR knockout CD8) + T cells).

[0086] (3) Lentiviral preparation Inoculate 1×10⁻⁶ cells into 6-well plates 6 / well 293T cells, and add 2ml of culture medium (basal medium containing 10% FBS + 4mM GlutaMax + 1mM sodium pyruvate, such as DMEM medium) to each well. After overnight incubation, retain 1ml of culture medium in each well.

[0087] Add 88.75 µl of reaction buffer (from the Xfect Transfection Reagent kit, catalog number 631318), psPASX plasmid (a prior art lentiviral packaging plasmid, 2.5 µg), pMD2.G plasmid (a prior art lentiviral envelope plasmid, 1.25 µg), and expression vector (5 µg) to EP tubes, and mix well. The expression vector is either R175H-TCR-pWPXL or R248Q-TCR-pWPXL expression vector. Then, add 2.625 µl of X-fect (from the Xfect Transfection Reagent kit, catalog number 631318) to the EP tube, mix well, and let stand at room temperature for 10 min. Add the above mixture to 293T cells. After 4 h, discard the supernatant and add 2 ml of fresh culture medium. Collect the supernatant after 48 hours, centrifuge at 300g for 8 minutes, and collect the supernatant again to obtain lentiviruses integrating R175H-TCR or R248Q-TCR, which will be used for TCRKO-CD8. + T cell transfection.

[0088] The R175H-TCR-pWPXL and R248Q-TCR-pWPXL expression vectors are formed by inserting gene fragments from either the R175H-TCR or R248Q-TCR into the empty pWPXL vector using conventional molecular biology techniques. pWPXL is a commonly used lentiviral shuttle vector and is commercially available.

[0089] The R175H-TCR gene fragment is specifically a T-cell receptor gene that targets the R175H mutant p53 protein antigen; the T-cell receptor protein it encodes (hereinafter referred to as the R175H-TCR protein) has the function of targeting the R175H mutant p53 protein. The sequence of the R175H-TCR gene fragment is (SEQ ID NO. 3).

[0090] The R248Q-TCR gene fragment is specifically a T-cell receptor gene that targets the R248Q mutant p53 protein antigen; the T-cell receptor protein it encodes (hereinafter referred to as the R248Q-TCR protein) has the function of targeting the R248Q mutant p53 protein. The sequence of the R248Q-TCR gene fragment is (SEQ ID NO. 4).

[0091] (4) Preparation of TCR-T cells co-expressing proteins targeting the R248Q / R175H mutant p53 protein antigen Using Dynabead superparamagnetic microbeads (Thermo Fisher Scientific), TCRKO-CD8 was activated at a Dynabead:cell ratio of 1:1. + T cells 24h.

[0092] Mix 300 µl of R175H-TCR lentivirus, 300 µl of R248Q-TCR lentivirus, and 100 µl of TMB medium containing 10 ng / ml IL-7 / 15 (interleukin-7 / 15). Infect magnetically activated TCRKO-CD8 cells with the above mixture. + T cells were infected for 12 hours, followed by supplementation with 700 µl of TMB medium containing 10 ng / ml IL-7 / 15. 24 hours post-infection, the supernatant was discarded, and 500 µl of 10 ng / ml IL-7 / 15 TMB medium was added. The medium was changed every 2 days to maintain a cell density of 5 × 10⁶ cells / mL. 5 Cells / ml. Six days after infection, the infection efficiency was assessed by flow cytometry, and TCR-T cells co-expressing R175H-TCR and R248Q-TCR were obtained. Figure 7 ).

[0093] Example 5: Preparation of bifunctional nanomedicines based on autophagy-targeting chimeric-T cell connectors In this technical solution, the bifunctional nanomedicine based on the autophagy-targeting chimera–T-cell connector (AUTACE) is implemented through the following process: TCR-T cells co-expressing R175H-TCR and R248Q-TCR proteins, prepared in Example 4, were used to extract their cell membranes for the preparation of nanomedicines. The cell membranes, incorporating both R175H-TCR and R248Q-TCR proteins, allow for the targeting of R175H and R248Q mutant p53 proteins. The cell membrane extraction process is as follows: Cells prepared in Example 4 were collected and centrifuged at 2000 rpm for 5 min to collect the precipitate. The collected precipitate was redispersed with PBS solution, and a conventional protease inhibitor was added to prevent protein degradation. The dispersed cell solution was homogenized, then centrifuged at 2000 rpm for 5 min to collect the supernatant. The precipitate was then redispersed with PBS, and the homogenization process was repeated. The precipitate was then collected by centrifugation at 12000 rpm for 20 min. The cell membranes collected in the two centrifugations were mixed (the supernatant from the first centrifugation + the precipitate from the second centrifugation) to obtain the cell membrane.

[0094] 26 mg SPC (soybean phosphatidylcholine), 1.5 mg cholesterol (Cholesterol, Chol), 1 mg DSPE-PEG2000 (distearate phosphatidylethanolamine-polyethylene glycol 2000), 2 mg KPY prepared in this method, and 4.5 mg PFP (perfluoropentane) were dissolved in 2 ml of chloroform and subjected to rotary evaporation using conventional methods. After rotary evaporation, a film was formed.

[0095] Then, 2 mL of double-distilled water and 3 mg of DSPE-PEG-streptavidin (distearate phosphatidylethanolamine-polyethylene glycol-streptavidin) were added to the membrane to hydrate it. After hydration, the membrane was subjected to ultrasonic treatment (under ice bath, frequency: 40 kHz, power: 180 W, time: 10 min, mode: 5 s on, 5 s off).

[0096] The ultrasonically treated mixture was then extruded using a conventional liposome extruder. After extrusion three times through a 100 nm polycarbonate membrane, drug-loaded avidin liposomes were obtained.

[0097] Liposomes and cell membranes were mixed at a mass ratio of 5:1, sonicated in an ice-water bath for 30 seconds (frequency: 40 kHz, power: 180 W), and then incubated at room temperature for 2 hours to achieve fusion, yielding KPPF@TCR liposomes. Biotinylated anti-CD3 antibody was added and mixed, and incubated at 4°C for 2 hours to achieve conjugation. The ratio of KPPF@TCR liposomes to biotinylated anti-CD3 antibody was 50:1. After the above treatment, the bifunctional nanomedicine based on autophagy-targeting chimeric T-cell connector (hereinafter referred to as AUTACE) was obtained. In the above synthesis process, except for cell membranes and KPY, all other reagents were commercially available. See the schematic diagram of the synthesis process. Figure 8 .

[0098] Nanoparticles that do not contain R175H-TCR, R248Q-TCR, KPY, and PFP are called T NVs. That is, in the preparation process, ordinary T cell membranes are used instead of the cell membranes of TCR-T cells that co-express R175H-TCR and R248Q-TCR, and KPY and PFP are not added. The nanomedicine is obtained by following the above procedure.

[0099] Transmission electron microscopy (TEM) shows that the bifunctional nanomedicine AUTACE prepared in this scheme exhibits a vesicle-like morphology. Figure 9 a). Its zeta potential is -16.98±1.24mV ( Figure 9 b), with a size of 153.27±28.35nm ( Figure 9 c), the particle concentration is approximately 1.2 × 10⁻⁶. 10 cells / mL ( Figure 9 d). Each particle carries approximately 1.5 × 10⁻⁶. 4 One anti-CD3 antibody ( Figure 9 e). The drug loading and encapsulation efficiency of KPY were 4.38% and 68.75%, respectively. KPY release was extremely low under baseline conditions, but LIFU irradiation triggered a rapid, "burst" release. Figure 9 f).

[0100] Example 6: In vitro antitumor effect of AUTACE Using AUTACE (1.2×10 10 / mL, 100μL), KPPF@TCR (1.3×10 10 / mL, 100μL) and T NV (1.2×10 10 PNAC-1 cells, MIA PaCa-2 cells, and SK-CO-1 cells were treated with 100 μL / mL for 6 h, followed by immunofluorescence and flow cytometry analysis. AUTACE (3 mg / mL) and AUTACE (3 mg / mL) + LIFU (1 W / cm²) were used. 2, 5min), AUTACE (3mg / ml) + LIFU (1W / cm 2 PNAC-1 cells and MIA PaCa-2 cells were treated with HCQ (10 μM) and KPY (20 μM) for 24 h for 5 min. Total cell protein was extracted and detected by Western blot.

[0101] Immunofluorescence and flow cytometry results showed that AUTACE could bind to PANC-1 and MIA PaCa-2 cells, but not to SK-CO-1 cells. Figure 10 ab); Western blot results showed that AUTACE, under LIFU irradiation, released KPY, which activated autophagy to degrade the mutant KRAS protein (ab); Figure 10 cd).

[0102] Subsequently, PANC-1 or MIA PaCa-2 tumor cells expressing luciferase were inoculated with human CD8. + T cells are distributed in a specific ratio (see ratio for details). Figure 11 Using T NV (3 mg / mL, KPPF@TCR (3 mg / mL), KPPF@TCR (3 mg / mL) + LIFU (1 W / cm) 2 , 5min), AUTACE (3mg / mL), AUTACE (3mg / mL) + LIFU (1W / cm 2 The AUTACE+LIFU group was treated with a mixture of tumor cells and T cells for 5 minutes. A control group containing only tumor cells and T cells (without any drug) and a control group treated with LIFU but without nanoparticles were also included. The tumor cell killing rate was statistically analyzed after the experiment. Compared with the control group, the AUTACE+LIFU group showed the strongest tumor cell killing effect when co-cultured with different nanoparticles (…). Figure 11 ab). Additionally, CD8 in the AUTACE or AUTACE+LIFU group + T cells exhibited enhanced activation and effector function, specifically manifested as increased CD69 expression and elevated levels of IFN-γ and GZMB. Figure 11 Further cell imaging results also confirmed that, compared with all controls, the AUTACE+LIFU group had the fewest residual tumor cells (cf). Figure 11 g).

[0103] In summary, AUTACE-based membrane-displayed TCRs recognize TP53 / KRAS co-mutant tumor cells; release KPY under LIFU triggering to achieve KRAS degradation; and recruit and activate CD8 cells via surface-anchored anti-CD3 receptors. + T cells thus produce an anti-tumor effect in TP53 / KRAS co-mutated tumors.

[0104] Example 7: Tumor Targeting of AUTACE To evaluate the in vivo tumor-targeting ability of AUTACE, mice carrying PANC-1 or MIA PaCa-2 tumors were intravenously injected with DiD-labeled nanoparticles at a concentration of 50 mg / kg (T NV and AUTACE). Tumor-specific signals from AUTACE were detected 2 hours post-injection and peaked at 6 hours; in contrast, T NV showed extremely low tumor enrichment. Figure 12 Furthermore, 48-hour ex vivo imaging showed that the tumor fluorescence intensity in the AUTACE group was higher than that in the TNV control group (ad). Figure 12 eh), while the two groups accumulated similarly in non-tumor organs ( Figure 12 Further fluorescence imaging of tumor sections also confirmed the enrichment of AUTACE in tumor tissue. Figure 12 In summary, AUTACE possesses excellent tumor-targeting properties and can simultaneously target TP53. R175H With TP53 R248Q Mutant tumor.

[0105] Example 8: In vivo antitumor effect of AUTACE The in vivo antitumor effect of AUTACE was evaluated in PANC-1 and MIA PaCa-2 subcutaneous xenograft mouse models. For the PANC-1 subcutaneous xenograft mouse model, the experimental groups were as follows: PBS group: PBS was injected into the tail vein of model mice on days 10, 14, 18 and 21.

[0106] T-only group: PBS was injected into the tail vein of the model mice on days 10, 14, 18, and 21; and CD8 was injected into the tail vein of the model mice on day 11. + T cells, dosage 1×10 7 On days 11, 12, and 13, IL-2 (interleukin-2) was injected into the tail vein of the model mice at a dose of 2 × 10⁻⁶. 5 U.

[0107] PFP@TCE group: On days 10, 14, 18 and 21, PFP@TCE was injected into the tail vein of model mice at a dose of 50 mg / kg. The difference between PFP@TCE and AUTACE is that PFP@TCE is not loaded with KPY, while all other aspects are the same as AUTACE.

[0108] PFP@TCE+LIFU group: On days 10, 14, 18, and 21, PFP@TCE was injected into the tail vein of model mice at a dose of 50 mg / kg; 6 hours after nanoparticle administration, mice were treated with LIFU at a parameter of 1 W / cm². 2 15min.

[0109] AUTACE group: On days 10, 14, 18 and 21, AUTACE was injected into the tail vein of model mice at a dose of 50 mg / kg.

[0110] AUTACE+LIFU group: On days 10, 14, 18, and 21, AUTACE was injected into the tail vein of model mice at a dose of 50 mg / kg; 6 hours after nanoparticle administration, the mice were treated with LIFU at a parameter of 1 W / cm². 2 15 min; In this group, LIFU promoted KPY release and initiated intracellular KRAS degradation, but because no additional CD8 was added... + T cells lead to the recruitment of CD8 by the T cell connector. + The function of T cells was not fully realized.

[0111] The PFP@TCE+T+LIFU group; on days 10, 14, 18, and 21, PFP@TCE was injected into the tail vein of model mice at a dose of 50 mg / kg; and LIFU treatment was applied to the mice 6 hours after nanoparticle administration, with LIFU parameters of 1 W / cm². 2 ,15min; and on day 11, CD8 was injected into the tail vein of the model mice. + T cells, dosage 1×10 7 On days 11, 12, and 13, IL-2 (interleukin-2) was injected into the tail vein of the model mice at a dose of 2 × 10⁻⁶. 5 U.

[0112] AUTACE+T+LIFU group: On days 10, 14, 18, and 21, AUTACE was injected into the tail vein of model mice at a dose of 50 mg / kg; 6 hours after nanoparticle administration, mice were treated with LIFU at a parameter of 1 W / cm². 2 ,15min; and on day 11, CD8 was injected into the tail vein of the model mice. + T cells, dosage 1×10 7 On days 11, 12, and 13, IL-2 (interleukin-2) was injected into the tail vein of the model mice at a dose of 2 × 10⁻⁶. 5 U.

[0113] For the MIA PaCa-2 subcutaneous xenograft mouse model, the experimental groups were as follows: PBS group: PBS was injected into the tail vein of the model mice on days 5, 9, 13 and 17.

[0114] T-only group: PBS was injected into the tail vein of model mice on days 5, 9, 13, and 17; and on day 6, CD8... + T-cell administration; and IL-2 administration on days 6, 7 and 8; the rest was the same as the PANC-1 experiment.

[0115] PFP@TCE group: PFP@TCE was administered on days 5, 9, 13 and 17; the rest was the same as in the PANC-1 experiment.

[0116] PFP@TCE+LIFU group: PFP@TCE was administered on days 5, 9, 13 and 17; the rest was the same as in the PANC-1 experiment.

[0117] AUTACE group: AUTACE dosage on days 5, 9, 13 and 17; otherwise the same as in the PANC-1 experiment.

[0118] AUTACE+LIFU group: AUTACE was administered on days 5, 9, 13 and 17; the rest was the same as in the PANC-1 experiment.

[0119] PFP@TCE+T+LIFU group; PFP@TCE was administered on days 5, 9, 13, and 17; and on day 6, CD8... + T-cell dosing; and on days 6, 7, and 8, the IL-2 dosing was 2 × 10⁻⁶. 5 U; the rest is the same as the PANC-1 experiment.

[0120] AUTACE+T+LIFU group: AUTACE was administered on days 5, 9, 13, and 17; and on day 6, CD8... + T-cell administration; and IL-2 administration on days 6, 7 and 8; the rest was the same as the PANC-1 experiment.

[0121] Compared with control groups such as PBS, PFP@TCE, AUTACE, PFP@TCE+LIFU, and T-only, AUTACE+LIFU significantly inhibited PANC-1 tumor growth; notably, combined with adoptive metastasis of CD8... + T cells (AUTACE+T+LIFU) further reduced tumor burden, and 4 out of 5 mice achieved complete tumor regression. Figure 13Immunohistochemistry showed decreased KRAS protein expression in tumor tissues of the AUTACE+LIFU and AUTACE+T+LIFU groups. Figure 13 d). Consistent with the results of PANC-1, a similar antitumor trend was also observed in the MIA PaCa-2 subcutaneous xenograft tumor model. Figure 13 e.g., AUTACE alone, under the action of LIFU, can achieve effective tumor growth inhibition. If AUTACE is combined with CD8... + T-cell and IL-2 therapy, combined with LIFU, can further inhibit tumor growth. AUTACE, CD8 + The dosage ratio of T-cell therapy to IL-2 therapy is 50 mg / kg × 4 : 1 × 10. 7 Quantity: 2×10 5 U×3, and 6 hours after nanoparticle administration, mice were treated with 1 W / cm 2 LIFU processing for 15 minutes.

[0122] Example 9: Biosafety of AUTACE After AUTACE treatment, the hematological parameters and serological indicators related to organ function in mice remained stable. Figure 14 a). Furthermore, histological examination of the heart, liver, spleen, lungs, kidneys, and intestines revealed no obvious signs of organ damage. Figure 14 b). This indicates that AUTACE has good biocompatibility.

[0123] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector, characterized in that, It includes a lipid shell; an anti-CD3 antibody is coupled to the lipid shell; and a T-cell receptor targeting mutant p53 protein is inserted into the lipid shell. The lipid shell encapsulates a drug KPY with the structural formula shown in Formula VII: Formula VII The lipid shell also encapsulates PFP; The bifunctional nanomedicine is obtained in the following manner: SS1: Obtaining a cell membrane containing a chimeric T-cell receptor targeting the mutant p53 protein; SS2: SPC, Chol, DSPE-PEG2000, KPY, and PFP were dissolved in chloroform and formed into a membrane by rotary evaporation; after hydration treatment, DSPE-PEG-streptavidin was added to the membrane; after ultrasonic treatment and liposome extrusion treatment, drug-loaded avidin liposomes were obtained. SS3: Drug-loaded avidin liposomes were mixed with cell membranes, and after sonication and incubation, KPPF@TCR liposomes were obtained. SS4: KPPF@TCR liposomes and biotinylated anti-CD3 antibody were mixed and incubated to obtain a bifunctional nanomedicine based on autophagy-targeting chimeric T-cell connector; the T-cell receptors targeting mutant p53 protein include T-cell receptor proteins with gene sequences as shown in SEQ ID NO. 3 for targeting R175H mutant p53 protein, and T-cell receptor proteins with gene sequences as shown in SEQ ID NO. 4 for targeting R248Q mutant p53 protein.

2. The bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector according to claim 1, characterized in that, The drug KPY is formed by linking KRB peptide with YOK-1304 via PEG2000; The structure of the KRB polypeptide is: Ac-KRRRRCPLYISYDPVCRRRRK(Biotin)-NH2; Ac represents the N-terminal acetyl group; the C-terminal carboxyl group of the KRB polypeptide is acetylated; the two cysteine ​​C residues in the KRB polypeptide are linked by a disulfide bond; Biotin indicates that the side chain amino group of lysine K is modified with biotin.

3. A bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector according to claim 2, characterized in that, The drug KPY was prepared by the following method: S1: Synthesis of YOK-NH2 YOK-1304 was reacted with BOC-6-aminohexanoic acid, EDC and DMAP in the solvent DMF; after the reaction was completed, the solvent was removed and the reaction was carried out in DCM containing TFA; after the reaction was completed, YOK-NH2 was obtained by purification and drying. S2: Synthesis of YOK-PEG2000-COOH YOK-NH2 was reacted with NHS-PEG2000-COOH and TEA in the solvent DCM; after the reaction was completed, YOK-PEG2000-COOH was obtained by purification and drying. S3: Synthesis of target drug KPY YOK-PEG2000-COOH was reacted with EDC and NHS in DMF solvent; after the reaction was completed and the solvent was removed, the product was dissolved in DCM solvent; after washing with water, drying and solvent removal, it was reacted with KRB peptide and TEA in DMF solvent; after the reaction was completed, the drug KPY was obtained after purification and drying.

4. A method for preparing a bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector according to any one of claims 1-3, characterized in that, The following steps are performed sequentially: SS1: Obtaining a cell membrane containing a chimeric T-cell receptor targeting the mutant p53 protein; SS2: SPC, Chol, DSPE-PEG2000, KPY, and PFP were dissolved in chloroform and formed into a membrane by rotary evaporation; after hydration treatment, DSPE-PEG-streptavidin was added to the membrane; after ultrasonic treatment and liposome extrusion treatment, drug-loaded avidin liposomes were obtained. SS3: Drug-loaded avidin liposomes were mixed with cell membranes, and after sonication and incubation, KPPF@TCR liposomes were obtained. SS4: KPPF@TCR liposomes and biotinylated anti-CD3 antibody were mixed and incubated to obtain a bifunctional nanomedicine based on autophagy-targeting chimeric T-cell connector.

5. A method for preparing a bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector according to claim 4, characterized in that, The cell membrane of a T cell receptor chimeric with a target mutant p53 protein was extracted from TCR-T cells containing membrane proteins with co-expressed gene sequences as shown in SEQ ID NO. 3 and membrane protein gene sequences as shown in SEQ ID NO.

4.

6. The method for preparing a bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector according to claim 5, characterized in that, The TCR-T cells were prepared by the following method: SSS1: CD8 + After T cells are activated by IL-2, the α and β chains of the cell's endogenous T cell receptor are knocked out, and CD8+ of the TCR is knocked out. + T cells; SSS2: The gene fragment with sequence such as SEQ ID NO. 3 and the gene fragment with sequence such as SEQ ID NO. 4 were integrated into the pWPXL expression vector to obtain the R175H-TCR-pWPXL expression vector and the R248Q-TCR-pWPXL expression vector, respectively. R175H-TCR lentivirus and R248Q-TCR lentivirus were constructed using the R175H-TCR-pWPXL expression vector and the R248Q-TCR-pWPXL expression vector, respectively. SSS3: TCR-T cells were obtained by co-infecting TCR-knockout CD8+ T cells with R175H-TCR lentivirus and R248Q-TCR lentivirus.

7. The application of a bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector according to any one of claims 1-3 in the preparation of a drug for treating KRAS / TP53 co-mutated tumors, characterized in that, The KRAS / TP53 comutated tumor is a KRAS-carrying tumor. G12D and TP53 R175H Double mutation, or carrying KRAS G12C and TP53 R248Q Double-mutated pancreatic cancer.

8. The application of the bifunctional nanomedicine based on an autophagy-targeting chimeric T-cell connector according to claim 7 in the preparation of products for treating KRAS / TP53 co-mutated tumors, characterized in that, Products for treating KRAS / TP53 comutated tumors include bifunctional nanomedicines based on autophagy-targeting chimeric-T cell connectors, devices for delivering LIFU, and CD8. + T cells and IL-2.

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