Preparation and application of polypeptide-drug conjugate based on msa-2 and hydrogel preparation thereof

Abstract

The present application relates to the preparation and application of MSA-2 based polypeptide-drug conjugate and hydrogel preparation thereof. The MSA-2 based polypeptide-drug conjugate is composed of MSA-2, self-assembling polypeptide and immune checkpoint blocking peptide, forming a multi-module structure of "MSA-2-self-assembling polypeptide-immune checkpoint blocking peptide"; it can self-assemble to form a nanofiber stable dispersion system in an aqueous medium, and the dispersion system can further form a hydrogel preparation under physiological conditions. The MSA-2 polypeptide drug conjugate hydrogel preparation loaded with immunotherapeutic agents can be prepared by blending and incubating the immunotherapeutic agents with the above nanofiber dispersion liquid. The present application realizes the self-delivery, long-acting slow release and local delivery of drugs by chemically coupling MSA-2 with self-assembling polypeptide to construct an injectable in-situ formed hydrogel, effectively solving the key problems of short half-life, high systemic toxicity and low lesion enrichment rate of MSA-2 in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide-drug conjugate based on the non-nucleotide-based STING activator MSA-2, a hydrogel formulation derived therefrom, its preparation method, and its applications. In particular, this invention covers the application of the hydrogel formulation in tumor immunotherapy, and its use as a highly efficient local delivery system for delivering immunotherapeutic agents such as oncolytic viruses, thereby overcoming existing delivery bottlenecks and enhancing their antitumor effects. Background Technology

[0002] Immunotherapy has become a cornerstone strategy in cancer treatment. Among them, the interferon gene stimulating factor (STING) pathway, as the core hub of innate immunity, can directly induce the production and release of type I interferon and pro-inflammatory factors when activated, and initiate anti-tumor immune responses and promote the activation of antigen-presenting cells, making it a highly promising therapeutic target.

[0003] MSA-2 is a novel, potent, and structurally stable non-nucleotide small molecule STING activator. Compared to cyclic dinucleotide agonists, it offers advantages such as relatively simple synthesis, good cell membrane permeability, and superior metabolic stability. Preclinical studies have demonstrated significant single-drug activity in various tumor models. Importantly, MSA-2 not only exhibits direct antitumor activity but also functions as a potent immune adjuvant. Studies have shown that MSA-2 effectively promotes the maturation of antigen-presenting cells and stimulates the generation and expansion of antigen-specific T cell responses. This characteristic gives it unique value in the development of therapeutic tumor vaccines, enabling it to synergistically interact with vaccine components and amplify the immune response by activating the STING pathway. Furthermore, the combination of MSA-2 with immune checkpoint blockade therapies (such as anti-PD-1 / PD-L1 antibodies) and other immunotherapies has been shown to produce significant synergistic antitumor effects, further broadening its clinical application prospects.

[0004] However, MSA-2 faces a key delivery bottleneck in clinical applications: Short half-life and insufficient accumulation in target tissues: MSA-2 has a short half-life in vivo, making it difficult to reach and maintain an effective therapeutic concentration at the tumor site after systemic administration, thus limiting its anti-tumor effect.

[0005] Significant systemic toxicity: Because the STING pathway is widely distributed in multiple cell types, systemic high doses of MSA-2 can trigger a strong systemic inflammatory response, leading to severe dose-limiting toxicity and limiting its therapeutic window.

[0006] Local immune microenvironment regulation requirements: As an immune adjuvant, the optimal effect of MSA-2 often requires continuous and controllable release in specific sites such as the tumor microenvironment or draining lymph nodes to maximize local anti-tumor immune activation while avoiding the side effects of non-target activation.

[0007] These delivery challenges severely limit the clinical translation potential of MSA-2. Therefore, developing delivery strategies that enable local, sustained, and controlled release of MSA-2 at target sites (such as within tumors) to reduce systemic exposure and toxicity while increasing local effective concentration and duration of action is a critical issue that urgently needs to be addressed.

[0008] It is noteworthy that MSA-2's potent immune adjuvant properties make it an ideal partner for synergistic delivery of various immunotherapies requiring local action, such as oncolytic viruses (OV), oncolytic bacteria, and CAR-T cells. For example, OV selectively kills tumor cells, releasing tumor antigens and danger signals to stimulate anti-tumor immunity. However, intratumoral injection of OV also faces challenges such as rapid clearance of viral particles, short local retention time, and insufficient persistence of immune activation. Similarly, other immunotherapies requiring local residence (such as CAR-T cells and oncolytic bacteria) also suffer from insufficient intratumoral persistence. Combining MSA-2 with these immunotherapies locally can produce a significant synergistic effect: immunotherapies (such as OV) mediate in situ tumor killing and antigen release, while locally sustained release of MSA-2 strongly amplifies DC activation and T cell responses, achieving a dual synergistic effect of "direct killing / antigen release" and "enhanced immune activation."

[0009] An ideal delivery system should possess the ability to prolong the local action time of drugs / immunotherapy agents, protect bioactive components (such as viruses and cells), and precisely regulate the tumor immune microenvironment, thereby significantly improving the efficacy and safety of combined immunotherapy.

[0010] Therefore, there is an urgent need to develop novel and efficient local delivery strategies that can achieve sustained and controlled release of the STING activator MSA-2 at the tumor site, reducing systemic exposure and toxicity, while simultaneously prolonging the retention and duration of action of immunomodulators such as OV within the tumor, and promoting the synergistic effect of precise delivery and immune activation. Summary of the Invention

[0011] This invention aims to overcome the key bottlenecks in the delivery of STING activator MSA-2 and its combined immunomodulators (such as oncolytic virus OV) in the prior art, and provides a peptide-drug conjugate based on MSA-2, an injectable hydrogel formulation derived therefrom, its preparation method, and its application in tumor immunotherapy.

[0012] This application constructs an injectable hydrogel formulation by chemically coupling MSA-2 with a self-assembled peptide, thereby achieving self-delivery, long-acting sustained release, and local delivery of the drug, effectively solving key problems in the prior art such as the short half-life of MSA-2, high systemic toxicity, and low lesion accumulation rate.

[0013] This formulation utilizes the degradation-responsive properties of hydrogels to not only prolong drug retention time at the tumor site and reduce dosing frequency, but also reduce systemic immune side effects through precise local delivery. Simultaneously, its three-dimensional porous structure facilitates the synergistic delivery of other immunotherapies (such as oncolytic viruses, OV). Furthermore, this self-assembled hydrogel formulation can also be coupled with other immune checkpoint blocking peptides for co-loading, enabling multi-pathway synergistic therapy and demonstrating broad application prospects in areas such as local treatment of solid tumors and postoperative adjuvant therapy.

[0014] The core component of this hydrogel formulation is the immunomodulatory molecule MSA-2 (STING activator), which is chemically coupled to an immune checkpoint blocking peptide containing a self-assembled polypeptide sequence via a linker to form an MSA-2-based polypeptide-drug conjugate.

[0015] This MSA-2-based peptide-drug conjugate can self-assemble into a gel matrix under physiological conditions and can be further incubated and integrated with immunomodulatory agents (such as OV). The formed gel system can serve as a local reservoir for immunomodulatory agents, enabling sustained release of the agents while synergistically releasing MSA-2 and immune checkpoint blocking peptides. This prolongs the intratumoral retention time of the immunomodulatory agents and enhances the induced immune response, effectively improving the antitumor effect.

[0016] The objective of this invention can be achieved through the following technical solutions: This invention first provides a peptide-drug conjugate based on MSA-2, which consists of MSA-2, a self-assembled peptide, and an immune checkpoint blocking peptide. The self-assembled polypeptide contains cysteine. Using a self-assembled polypeptide as a backbone, the sulfhydryl group of the cysteine ​​side chain in the self-assembled polypeptide is coupled to MSA-2 via a linker that can be cleaved by esterases. The C-terminus of the self-assembled polypeptide sequence is linked to an immune checkpoint blocking peptide. The MSA-2-based peptide-drug conjugate forms a multi-module linear structure of “MSA-2–self-assembled peptide–immune checkpoint blocking peptide”. The MSA-2-based peptide-drug conjugate can self-assemble in an aqueous medium to form a stable dispersion system with nanofibers as the basic unit. Under physiological conditions, the dispersion system can further form a functionalized hydrogel formulation.

[0017] In some embodiments of the present invention, the immune checkpoint blocking peptide includes, but is not limited to, one or a combination of several of PD-1 / PD-L1 blocking peptides and CD47 / SIRPα blocking peptides.

[0018] Optionally, the PD-1 / PD-L1 blocking peptide can be selected as the polypeptide SGQYASYHCWCWRDPGRSGGSK or NYSKPTDRQYHF.

[0019] In some embodiments of the present invention, the self-assembling polypeptide includes, but is not limited to, one or a combination of several of CVVA, CVVVA, CVVAA, CVVVAAA, CVVGRGD, CRGD, GFFY, GFFYY, GFFYGRGD, GFFYYGKRGD, KFKFEFKFE, FEFEFKFK, or RADARADARADA.

[0020] In one embodiment of the present invention, the MSA-2-based peptide-drug conjugate is specifically MSA- D PPA1 is composed of the STING activator MSA-2 prodrug molecule, a self-assembled polypeptide, and a PD-L1 antagonist peptide. D PPA1 is constructed by sequential coupling, where, D PPA1 refers to D-type peptide. D PPA-1, also written as (D)-PPA 1, CAS No.: 1620813-53-7, has the following structure: .

[0021] The present invention further provides a method for preparing a peptide-drug conjugate based on MSA-2, the specific steps of which are as follows: S1. Covalently couple MSA-2 with a linker to obtain the MSA-2 prodrug molecule; S2. The MSA-2 prodrug molecule obtained in S1 is conjugated with a self-assembled polypeptide and an immune checkpoint blocking peptide via a cysteine ​​side chain thiol group to prepare an MSA-2-based polypeptide-drug conjugate.

[0022] In one embodiment of the present invention, when the MSA-2-based peptide-drug conjugate is specifically MSA- D At PPA1, MSA- D The preparation method of PPA1 is as follows: S1. Couple MSA-2 with the linker SS-py to obtain the MSA-2 prodrug molecule; S2, Combine the MSA-2 prodrug molecule obtained in S1 with... D PPA1 self-assembled peptide conjugation yielded the peptide-drug conjugate MSA-2.D PPA1.

[0023] In one embodiment of the present invention, the structure of the connector SS-py in step S1 is as follows: .

[0024] In one embodiment of the present invention, the structure of the MSA-2 prodrug molecule in step S1 is shown below: .

[0025] The present invention further provides a hydrogel formulation of a peptide-drug conjugate based on MSA-2, which is made by dissolving the peptide-drug conjugate based on MSA-2 in an aqueous solvent and allowing it to self-assemble into a nanofiber dispersion system. The nanofiber dispersion system can form a porous network structure hydrogel formulation under physiological conditions.

[0026] In some embodiments of the present invention, the hydrogel formulation may further encapsulate one or more immunotherapeutic agents.

[0027] In some embodiments of the present invention, the immunotherapeutic agent includes, but is not limited to, one or a combination of oncolytic viruses, oncolytic bacteria, CAR-engineered immune cells, immune checkpoint inhibitors, or cytokines.

[0028] In some embodiments of the present invention, the oncolytic virus includes, but is not limited to, one or more of the following: adenovirus, herpes simplex virus, vaccinia virus, echovirus, reovirus, and measles virus.

[0029] In some embodiments of the present invention, the oncolytic bacteria include, but are not limited to, one or more of the following: attenuated Salmonella, Clostridium, Listeria, Escherichia coli, or Bifidobacterium.

[0030] In some embodiments of the present invention, the CAR-engineered immune cells include, but are not limited to, one or more of the following: CAR-T cells, CAR-NK cells, and CAR-macrophages.

[0031] In some embodiments of the present invention, the immune checkpoint blocker includes, but is not limited to, one or more of the following: PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, TIM-3 antibody, LAG-3 antibody, and CD47 antibody.

[0032] In some embodiments of the present invention, the cytokines include, but are not limited to, one or more of the following: IL-2, IL-15, IFN-γ, TNF-β, CXCL-9 / 10 / 11, IFN-α / β, CCL27 / 28, granulocyte-macrophage colony-stimulating factor (GM-CSF), and Fms-like tyrosine kinase 3 ligand (Flt3L).

[0033] Preferably, the immunotherapeutic agent is selected as oncolytic adenovirus (OAds).

[0034] This invention further provides a method for preparing a hydrogel formulation of a peptide-drug conjugate based on MSA-2, comprising the following steps: The MSA-2-based peptide-drug conjugate was dissolved in an aqueous medium and allowed to stand at room temperature to induce self-assembly and form a nanofiber dispersion system. This nanofiber dispersion system can transform in situ into a hydrogel under physiological conditions.

[0035] When a hydrogel formulation is loaded with an immunotherapeutic agent, the preparation method of the hydrogel formulation loaded with the immunotherapeutic agent (also known as an immunomodulatory hydrogel formulation) includes the following steps: The MSA-2-based peptide-drug conjugate was dissolved in an aqueous medium and allowed to stand at room temperature to induce self-assembly and form a nanofiber dispersion system. Immunotherapy agents were incorporated into the obtained nanofiber dispersion system, and after gentle mixing, an immunomodulatory hydrogel formulation loaded with immunotherapy agents was obtained.

[0036] In one embodiment of the present invention, the aqueous medium is selected from either deionized water or phosphate buffer; and is left to stand at room temperature for 0.5-5 days.

[0037] This invention further provides the use of MSA-2-based peptide-drug conjugate hydrogel formulations or immunomodulatory hydrogel formulations loaded with immunotherapeutic agents in the preparation of antitumor or cancer drugs, enabling local controlled-release delivery of immunotherapeutic agents. After local injection, it can form a hydrogel under physiological conditions.

[0038] The immunomodulators include, but are not limited to, one or a combination of oncolytic viruses, oncolytic bacteria, CAR-engineered immune cells, immune checkpoint inhibitors, or cytokines.

[0039] In one embodiment of the present invention, preferably, the immunotherapeutic agent is an oncolytic adenovirus (OAds).

[0040] When the immunomodulator is an oncolytic virus, it can be used for local delivery of the oncolytic virus. After intratumoral injection or postoperative tumor cavity filling, the formulation achieves effective local delivery and retention of the oncolytic virus, preventing its rapid systemic clearance; the released MSA-2 activates the STING pathway; and the synergistically released immune checkpoint blocking peptide works in conjunction with the oncolytic virus and the activated immune pathway to coordinate and enhance antitumor efficacy, thereby significantly improving the therapeutic effect based on oncolytic viruses.

[0041] In one embodiment of the present invention, OAds are inoculated into an immunomodulatory hydrogel formulation for OAds delivery (i.e., a hydrogel formulation of a peptide-drug conjugate based on MSA-2), and incubated to obtain an OAds-loaded immunomodulatory hydrogel formulation. This OAds-loaded immunomodulatory hydrogel formulation can transform into a hydrogel state under physiological conditions.

[0042] In one embodiment of the present invention, the incubation temperature is 37°C and the incubation time is 30 minutes.

[0043] Immunomodulatory hydrogel formulations loaded with OAds can be used directly as antitumor or cancer drugs, and can be converted into a hydrogel state under physiological conditions when used.

[0044] In one embodiment of the present invention, the immunomodulatory hydrogel formulation loaded with OAds can be used directly as an antitumor or cancer drug, and it can undergo in-situ gelation under physiological conditions.

[0045] In one embodiment of the present invention, the antitumor or cancer drug is a drug that inhibits tumor growth, recurrence and metastasis and prolongs the user's survival period.

[0046] In one embodiment of the present invention, the antitumor or cancer drug is a drug that can promote the infiltration of lymphocytes in the tumor microenvironment, thereby effectively inhibiting tumor growth. OAds are delivered locally through this hydrogel system and exert a sustained antitumor effect.

[0047] In one embodiment of the present invention, the tumor or cancer includes, but is not limited to, one or more of melanoma, breast cancer, lymphoma, hepatocellular carcinoma, prostate cancer, lung cancer, glioma, pancreatic cancer, sarcoma, head and neck cancer, cervical cancer, endocrine tumor, adenoma, colon cancer, rectal cancer, blastoma, gastric cancer, or esophageal cancer.

[0048] The meanings of the polypeptide sequences in this invention are explained as follows: NYSKPTDRQYHF refers to a polypeptide with the amino acid sequence NYSKPTDRQYHF, CVVAA refers to a polypeptide with the amino acid sequence CVVAA, and so on. Wherein, amino acid C represents cysteine, V represents valine, and A represents alanine.

[0049] This invention provides a peptide-drug conjugate based on MSA-2 and an immunomodulatory hydrogel formulation. The formulation is administered as an injectable nanofiber solution, rapidly forming a three-dimensional network gel at the lesion site to construct a local drug reservoir. The system is composed of biocompatible and biodegradable materials, achieving highly efficient co-delivery of the STING activator (MSA-2) and immunomodulatory agents (such as oncolytic viruses) without the need for exogenous carriers. Through precise local delivery, the hydrogel can sustainably release MSA-2 and immune checkpoint blocking peptides (such as... D PPA1) enables sustained immune activation.

[0050] The MSA-2-based polypeptide-drug conjugate hydrogel formulation of this invention, when administered topically, can significantly enhance the retention and infiltration of immunomodulators (such as oncolytic viruses) at tumor sites, and achieve the interaction between the immunomodulatory molecule MSA-2 and immune checkpoint blocking peptides (such as... D Controlled release of PPA1).

[0051] In the preferred embodiment of this application, the immunomodulator is selected as oncolytic adenovirus OAds, and the immune checkpoint blocking peptide is selected as... D When PPA1 is used, this formulation can enhance local drug concentration and bioavailability while exerting a synergistic effect between OAds and immunomodulatory molecules. Specifically, the STING activator MSA-2 promotes antigen processing and presentation by activating the STING signaling pathway, enhances dendritic cell activation, and thus significantly increases CD8 levels. + T cell activation efficiency; D PPA1 can block tumor PD-1 / PD-L1-mediated immune escape and promote CD8. + T cells specifically recognize and kill tumors, effectively inhibiting tumor progression and significantly prolonging survival. Furthermore, the OAds-loaded immunomodulatory hydrogel formulation of this invention can further stimulate the body's systemic immune response, induce long-lasting anti-tumor immune memory, and effectively inhibit tumor recurrence.

[0052] Chinese patent CN119219600B discloses a water-soluble prodrug compound of the STING agonist MSA-2, its preparation method, and its applications. The provided water-soluble prodrug compound of STING agonist MSA-2 is formed by linking an alkanolamine molecule with MSA-2 via an ester group, significantly improving its water solubility. After entering the body, this prodrug can be hydrolyzed by esterases to release the original drug MSA-2 for cancer treatment. The water-soluble MSA-2 prodrug exhibits excellent water solubility, supporting intravenous administration, thereby increasing blood drug concentration, reducing dosage, and lowering the risk of adverse reactions.

[0053] Although the above methods have confirmed that the modified MSA-2 has excellent water solubility and can increase blood concentration, reduce drug dosage and reduce adverse reactions through intravenous administration, the core of the modification focuses on the MSA-2 molecule itself, and the route of administration still mainly relies on intravenous injection. The delivery strategy has not been optimized, nor has it been specifically designed for the duration of drug action.

[0054] Chinese patent CN118236513A discloses a paclitaxel immunomodulatory hydrogel formulation, its preparation method, and its applications. The core of this formulation is a nanofiber solution self-assembled from a conjugate formed by paclitaxel and the targeting peptide iRGD, with immunotherapeutic agents (such as cytokines and STING agonists) added to this solution. This hydrogel can act as a drug reservoir, enabling slow drug release. After a single dose, it not only directly inhibits tumors but also activates a long-lasting in vivo immune response, significantly increasing lymphocyte infiltration in the tumor microenvironment, thereby effectively inhibiting tumor growth, recurrence, and metastasis.

[0055] Although the above-mentioned approach has demonstrated that adding immunotherapeutic agents to peptide-drug conjugate hydrogels can achieve sustained-release effects, its core is to extend the effect of immunotherapeutic agents by constructing an in-situ drug reservoir based on paclitaxel conjugates. It does not specifically construct a peptide-drug conjugate gel formulation system for the immunotherapeutic agents themselves, nor does it integrate immune checkpoint blocking peptides and drug molecules through drug conjugate design strategies to achieve synergistic effects.

[0056] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Innovative Self-Delivery and Controlled Release System: The core of this invention lies in the use of MSA-2-based peptide-drug conjugate gels, which achieve a breakthrough in the self-assembly of hydrogel drug reservoirs without the need for exogenous carriers. This not only completely avoids the biocompatibility risks of traditional carrier materials, but also achieves controllable management of drug release kinetics through precise regulation of molecular structure, effectively solving the common burst release problem in traditional formulations. The core advantage of this modular design is that it allows for the integration of immune checkpoint blocking peptides. D PPA-1 or other immune checkpoint blocking peptides provide a highly flexible technical platform for constructing synergistic therapeutic systems.

[0057] (2) Long-lasting immune activation and synergistic mechanism: Thanks to the sustained-release properties of MSA-2's peptide-drug conjugate gel, an effective drug concentration within the therapeutic window can be maintained at the lesion site, thereby achieving long-term and stable activation of the STING pathway. This mechanism significantly overcomes the short-lived effect of traditional STING agonists, greatly improving the activation efficiency of antigen-presenting cells and prolonging the duration of the immune response. More importantly, this design incorporates immune checkpoint blockade functions (such as... DPPA-1's immune checkpoint blockade not only activates innate immunity but also effectively alleviates T cell depletion and enhances its anti-tumor activity, achieving a deep-level synergistic immune enhancement.

[0058] (3) Precise local delivery, synergistic therapeutic expansion, and enhanced safety: Employing a local injection strategy, this system significantly improves drug retention rate and target concentration at the tumor microenvironment. Its inherent peptide-drug conjugate structure enables efficient co-delivery of STING agonists and immune checkpoint blocking peptides (such as...). D The platform possesses the capability to deliver PPA-1. More importantly, it can be further expanded to locally load other immunotherapeutic agents (such as oncolytic viruses). This co-delivery and loading strategy ensures that multiple therapeutic components (STING agonists, immune checkpoint blocking peptides, oncolytic viruses, etc.) work synergistically in the lesion space and time, significantly extending the therapeutic window and achieving deep synergistic effects. Furthermore, the entire system is constructed based on biocompatible, biodegradable peptide materials, combined with precise local drug delivery, minimizing systemic drug exposure, effectively avoiding related systemic toxicity, and significantly improving the overall safety of treatment. Attached Figure Description

[0059] Figure 1 MSA, the polypeptide-drug conjugate of Example 1 of this invention. D Chemical structural formula of PPA1.

[0060] Figure 2 MSA, the polypeptide-drug conjugate of Example 1 of this invention. D Mass spectrometry analysis results of PPA1.

[0061] Figure 3 This is a transmission electron microscope (TEM) characterization image of the MDF in Example 2 of the present invention.

[0062] Figure 4 This is a characterization diagram of the "solution-gel" transition of MDF in Example 2 of the present invention.

[0063] Figure 5 This is a rheological test diagram of the "solution-gel" transformation process of MDF in Example 2 of the present invention.

[0064] Figure 6 This is a diagram showing the drug release characteristics of MDF-Gel in Example 2 of the present invention.

[0065] Figure 7 This is a scanning electron microscope (SEM) image of the MDF-Gel gel after lyophilization in Example 2 of this invention.

[0066] Figure 8 This is a three-dimensional confocal image of OAds / MDF-Gel in Embodiment 3 of the present invention.

[0067] Figure 9 In Example 4 of this invention, after treatment with OADs / MDF-Gel, intratumoral CD8... + T cell infiltration level.

[0068] Figure 10 In Example 4 of this invention, after treatment with OADs / MDF-Gel, CD80 / 86 levels in lymph nodes were observed. + Cell infiltration level.

[0069] Figure 11 This is an H&E stained section image of a mouse brain tumor after treatment with OAds / MDF-Gel, as shown in Example 5 of the present invention.

[0070] Figure 12 This is the survival curve of mice after treatment with OADs / MDF-Gel in Example 5 of the present invention. Detailed Implementation

[0071] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should be emphasized that the specific embodiments described are intended to illustrate the present invention and not to limit it.

[0072] The sources of the required materials in the following embodiments are explained as follows: MSA-2 was purchased from Maclean's, and all amino acids were purchased from Shanghai Jier Biochemical Co., Ltd.

[0073] Unless otherwise specified, other reagents or materials are all commercially available products in the field, and their processing techniques are conventional techniques in the field.

[0074] The present invention will be further illustrated by specific embodiments below. It should be understood that these embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the present invention.

[0075] Example 1 MSA-2-based peptide-drug conjugates MSA- D Preparation of PPA1 3.03 g of 2,2'-dithiopyridine was dissolved in 20 mL of methanol to obtain a reaction solution. Then, 1 g of 3-mercapto-1-propanol was weighed and dissolved in 10 mL of methanol, and this solution was gradually added dropwise to the reaction solution while stirring for 6 hours. Finally, the reaction solution was purified by column chromatography to obtain the linker SS-py. 100 mg of MSA-2 was dissolved in 50 mL of anhydrous dichloromethane, followed by the addition of 129.4 mg of EDCI, 8.25 mg of DMAP, and 74.7 mg of SS-py. The reaction solution was stirred under nitrogen protection for 48 hours, and finally purified by column chromatography to obtain the MSA-2 prodrug molecule.

[0076] D PPA1 modified peptide (sequence: N-CVVAAPLGLAG(NYSKPTDRQYHF)) D -C) was prepared by the standard Fmoc solid-phase synthesis method and then acetylated to obtain the final product.

[0077] The above MSA-2 prodrug molecule and D The PPA1-modified peptide was dissolved in 1 mL of dimethyl sulfoxide (DMSO) at a 1:2 molar ratio, and the reaction was carried out under nitrogen protection at room temperature with stirring for 36 hours to finally obtain the MSA-2 peptide-drug conjugate MSA- D PPA1. MSA- D PPA1 is dissolved in deionized water and allowed to stand at room temperature for 24 hours to obtain nanofiber solution MDF.

[0078] Figure 1 The MSA-2 polypeptide-drug conjugate in this invention is MSA- D The chemical structural formula of PPA1 and its mass spectrometry analysis results are as follows: Figure 2 As shown, this confirms MSA- D Successful synthesis of PPA1.

[0079] Example 2 MSA- D Preparation and characterization of PPA1 nanofiber solution MDF and immunomodulatory hydrogel MDF-Gel MSA- D The preparation methods of PPA1 nanofiber solution MDF and immunomodulatory hydrogel MDF-Gel are as follows: MSA- D PPA1 was dissolved in deionized water and allowed to stand at room temperature for 24 hours to obtain an MDF fiber solution. Figure 3 The image shows a transmission electron microscope (TEM) image of the MDF nanofibers, demonstrating the successful preparation of the MDF nanofibers.

[0080] like Figure 4 The inverted bottle experiment shown demonstrates that the MSA- in this invention D PPA1 nanofiber (MDF) solution can be transformed from a solution state to a gel state under physiological conditions simulated by the addition of PBS.

[0081] like Figure 5 The rheological tests shown indicate that the system exhibits G' > G", confirming the formation of MDF-Gel.

[0082] 20 μL of 10×PBS was added to 180 μL of 5 mM MDF solution to form MDF-Gel. The solution was then incubated at 37°C with 1 mL of pH 7.4 PBS. During a 30-day experimental period, the supernatant was collected at predetermined time points and replenished with an equal volume of PBS. MSA- was detected and analyzed using high-performance liquid chromatography (HPLC). D Cumulative release of PPA1.

[0083] like Figure 6 As shown, the hydrogel drug reservoir formed by MDF (MDF-Gel) can achieve MSA- in vitro. D The PPA1 drug molecule is continuously released for up to 30 days, with a cumulative release rate of 56.46%.

[0084] like Figure 7 As shown, the MDF-Gel formed by the nanofiber solution exhibits a three-dimensional porous network structure, providing ample physical space for the loading of subsequent immunomodulators.

[0085] Example 3 MSA-2 peptide-drug conjugate gel (MDF-Gel) loaded with oncolytic adenovirus (OAds) The method for loading oncolytic viruses onto MDF-Gel is as follows: OAds were added to 180 μL of MDF solution and incubated at 37°C for 30 minutes. Then, 20 μL of 10× PBS was added to obtain OAds-MDF hydrogel (OAds-MDF-Gel).

[0086] To facilitate characterization of the distribution of oncolytic viruses in the gel, FITC-labeled OAds were used.

[0087] like Figure 8 As shown in the confocal 3D image, the green fluorescent signal represents FITC-labeled OAds, and the results confirm that the OAds are uniformly dispersed in the MDF-Gel, thus confirming the successful construction of OAds-MDF-Gel.

[0088] Example 4 MSA-2 peptide-drug conjugate gel (MDF-Gel) for local delivery of oncolytic adenovirus OADs, enhancing oncolytic virus therapy and immune activation. First, C57BL / 6 mice were subcutaneously injected with 2×10⁻⁶ mg / kg for 6-8 weeks. 6 A subcutaneous melanoma model was constructed using B16-OVA. After modeling, mice were randomly divided into the following groups: (i) Saline treatment group, (ii) free OAds treatment group (1*10). 9PFU); and (iii) OAds-MDF-Gel treatment group (MSA-2 10mg / kg, D PPA1 52.8mg / kg, OAds 1*10 9 PFU (50 μL per animal) was injected locally into the tumor. Subsequently, on day 7 post-administration, tumor tissue was collected, and single-cell suspensions were obtained by enzymatic digestion. Inguinal lymph nodes were also collected and ground to obtain single-cell suspensions, which were further analyzed by flow cytometry to determine the infiltration of immune cells.

[0089] Figure 9 CD8 was displayed + The proportion of T cells in tumor tissue showed that OAds-MDF-Gel significantly enhanced CD8. + T cell activation was also observed, and the results were superior to those of the free OAds experimental group.

[0090] Figure 10 CD80 / 86 was displayed. + The proportion of cells in lymph node DC cells was measured, and the results showed that OAds-MDF-Gel significantly enhanced the activation of DC cells in lymph nodes.

[0091] Example 5 MSA-2 peptide-drug conjugate gel (MDF-Gel) for local delivery of oncolytic adenoviruses (OAds) inhibits postoperative tumor recurrence and prolongs survival in mice. C57BL / 6 mice were anesthetized and fixed on a small animal stereotaxic apparatus. After disinfecting the scalp, the skin was incised along the sagittal suture to fully expose the skull surface. Using the anterior fontanelle as an anatomical landmark, the target coordinates were located 1 mm lateral to the right and 2 mm anteriorly, and a fine hole was drilled using a micro-drill. Subsequently, a micro-injector was inserted vertically to a depth of 2.5 mm. 5 μL of GL261 glioma cell suspension (total 2 × 10⁻⁶ cells) was slowly injected at a rate of 0.5 μL / min. 5 (Number of gliomas). After injection, the needle was kept in place for 5 minutes to prevent backflow of fluid, and then withdrawn at a speed of 1 mm / min. Finally, the skull hole was sealed with sterile bone wax, and the skin incision was sutured layer by layer to complete the construction of the in situ glioma model.

[0092] Tumor resection was performed on day 7 after modeling, and OAds-MDF solution (MSA-210 mg / kg) was administered. D PPA 152.8 mg / kg, OAds 1*10 7 PFU (5 μL) was injected into the postoperative resection cavity. Simultaneously, saline and FreeOAds treatment groups were set up as controls.

[0093] Figure 11 shows the results of hematoxylin and eosin (H&E) staining of mouse brain sections, indicating that, compared with the saline treatment group and the free oncolytic adenovirus experimental group (Free OAds), the gliomas in the OAds-MDF-Gel treatment group did not show significant recurrence.

[0094] The survival curves shown in Figure 12 indicate that mice treated with OAds-MDF-Gel had significantly longer survival times compared to the saline-treated group and the Free OAds experimental group.

[0095] The embodiments described herein are intended to help those skilled in the art quickly understand and implement this technical solution. For those skilled in the art, based on the core ideas of this invention, the embodiments can be flexibly adjusted, or its principles can be applied to other similar scenarios without additional innovative work.

[0096] Therefore, the scope of protection of this invention is not limited to the specific embodiments described in the specification. Any reasonable modifications, equivalent substitutions, or optimizations made based on the concept of this invention are within the scope of protection of this patent.

Claims

1. A peptide-drug conjugate based on MSA-2, characterized in that, It is composed of MSA-2, self-assembling peptides, and immune checkpoint blocking peptides. The self-assembled polypeptide contains cysteine. Using a self-assembled polypeptide as a backbone, the sulfhydryl group of the cysteine ​​side chain in the self-assembled polypeptide is coupled to MSA-2 via a linker that can be cleaved by esterases. The C-terminus of the self-assembled polypeptide sequence can be linked to an immune checkpoint blocking peptide. The MSA-2-based peptide-drug conjugate forms a multi-module structure of "MSA-2 – self-assembled peptide – immune checkpoint blocking peptide"; The MSA-2-based peptide-drug conjugate can self-assemble in an aqueous medium to form a stable nanofiber dispersion system, which can further form a hydrogel formulation under physiological conditions.

2. The MSA-2-based peptide-drug conjugate according to claim 1, characterized in that, The immune checkpoint blocking peptides include, but are not limited to, one or a combination of several of PD-1 / PD-L1 blocking peptides and CD47 / SIRPα blocking peptides. Preferably, the PD-1 / PD-L1 blocking peptide is selected as the polypeptide SGQYASYHCWCWRDPGRSGGSK or NYSKPTDRQYHF.

3. The MSA-2-based polypeptide-drug conjugate according to claim 1, characterized in that, The self-assembling peptides include, but are not limited to, one or a combination of several of CVVA, CVVVA, CVVAA, CVVVAAA, CVVGRGD, CRGD, GFFY, GFFYY, GFFYGRGD, GFFYYGKRGD, KFKFEFKFE, FEFEFKFK, or RADARADARADA.

4. The MSA-2-based polypeptide-drug conjugate according to claim 1, characterized in that, The specific MSA-2-based peptide-drug conjugate is MSA- D PPA1 is composed of the STING activator MSA-2 prodrug molecule, a self-assembled polypeptide, and a PD-L1 antagonist peptide. D PPA1 is constructed by sequential coupling, where, D The structure of PPA1 is as follows: 。 5. A hydrogel formulation of a peptide-drug conjugate based on MSA-2, characterized in that, It involves dissolving the MSA-2-based peptide-drug conjugate as described in any one of claims 1-4 in an aqueous solvent, allowing it to self-assemble into a nanofiber dispersion system. The nanofiber dispersion system can form a porous network structure hydrogel under physiological conditions.

6. A hydrogel formulation of a peptide-drug conjugate based on MSA-2 according to claim 5, characterized in that, The hydrogel formulation encapsulates one or more immunotherapeutic agents; The immunotherapeutic agents include, but are not limited to, one or a combination of several of oncolytic viruses, oncolytic bacteria, chimeric antigen receptor engineered immune cells, immune checkpoint blockers, or cytokines.

7. A hydrogel formulation of a peptide-drug conjugate based on MSA-2 according to claim 6, characterized in that, The oncolytic virus is selected from one or more of the following: adenovirus, herpes simplex virus, vaccinia virus, echovirus, reovirus, measles virus; The oncolytic bacteria are selected from one or more of the following: attenuated Salmonella, Clostridium, Listeria, Escherichia coli, or Bifidobacterium; The CAR-engineered immune cells are selected from one or more of the following: CAR-T cells, CAR-NK cells, and CAR-macrophages; The immune checkpoint inhibitor is selected from one or more of the following: PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, TIM-3 antibody, LAG-3 antibody, CD47 antibody; The cytokines are selected from one or more of the following: IL-2, IL-15, IFN-γ, TNF-β, CXCL-9 / 10 / 11, IFN-α / β, CCL27 / 28, granulocyte-macrophage colony-stimulating factor, and Fms-like tyrosine kinase 3 ligand.

8. The use of the hydrogel formulation of the MSA-2-based peptide-drug conjugate according to any one of claims 5 to 6 in the preparation of antitumor or cancer drugs, characterized in that, Used to achieve local controlled-release delivery of immunotherapeutic agents, which can form a hydrogel under physiological conditions after local injection.

9. The application according to claim 8, characterized in that, The drug is suitable for inhibiting tumor progression, preventing recurrence and metastasis, and prolonging patient survival.

10. The application according to claim 8, characterized in that, The tumors or cancers mentioned include, but are not limited to, one or more of the following: melanoma, breast cancer, lymphoma, hepatocellular carcinoma, prostate cancer, lung cancer, glioma, pancreatic cancer, sarcoma, head and neck cancer, cervical cancer, endocrine tumors, colon cancer, nephroblastoma, germ cell tumors, rectal cancer, blastoma, gastric cancer, or esophageal cancer.

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