NLG919-polypeptide drug conjugate and preparation method and application of hydrogel preparation of NLG919-polypeptide drug conjugate

Abstract

The invention relates to an NLG919-polypeptide drug conjugate and a preparation method and application of a hydrogel preparation of the NLG919-polypeptide drug conjugate. The NLG919-polypeptide drug conjugate is composed of NLG919, a self-assembly polypeptide and an immune function polypeptide, and a multi-segment structure of the NLG919-self-assembly polypeptide-immune function polypeptide is formed. In an aqueous solvent, the NLG919-polypeptide drug conjugate can be self-assembled into nanofibers, and a three-dimensional porous immunoregulatory hydrogel scaffold is formed under physiological conditions. The hydrogel system has good injectability, and can continuously release NLG919 and immune function polypeptide as a local drug reservoir after administration, so as to regulate and control a tumor immunosuppression microenvironment. The hydrogel preparation is suitable for delivering adoptive immune cells represented by CAR-T cells, in-situ embedding and sustained cell release can be realized in tumor lesions or postoperative resection cavities, local retention time is remarkably prolonged, and intra-tumor infiltration and effector functions are enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the preparation method and application of NLG919-peptide drug conjugates and their hydrogel formulations. Specifically, this application provides an NLG919-peptide drug conjugate based on the immunomodulatory molecule NLG919, its preparation method, an immunomodulatory hydrogel formulation formed by the self-assembly of nanofibers from this conjugate, and the application of the immunomodulatory hydrogel formulation in tumor immunotherapy, particularly in the regulation of the tumor immune microenvironment and local delivery of adoptive immune cells (such as CAR-T cells). Background Technology

[0002] Tumor immunotherapy, by activating or rebuilding the body's own immune system to specifically recognize and eliminate tumor cells, has become the fourth major cancer treatment strategy after surgery, radiotherapy, and chemotherapy. In recent years, various immunotherapies, represented by immune checkpoint inhibitors, adoptive immunotherapy, and tumor vaccines, have made significant progress in relapsed / refractory malignancies. Among them, adoptive immunotherapy (such as chimeric antigen receptor T (CAR-T) cell therapy) has achieved clinical translation in hematologic malignancies such as B-cell leukemia and lymphoma, with some patients achieving long-term remission or even clinical cure. However, in the treatment of solid tumors, the presence of a suppressive tumor immune microenvironment (TIME) still significantly limits the overall efficacy of adoptive immunotherapy.

[0003] Current research indicates that tumor immunosuppression not only depends on immune checkpoint signaling pathways such as PD-1 / PD-L1, but is also closely related to tumor-associated immunometabolic reprogramming. Indoleamine-2,3-dioxygenase 1 (IDO1) is one of the key regulatory enzymes of tumor immunometabolic suppression. IDO1 catalyzes the metabolism of tryptophan along the kynurenine (Kyn) pathway, leading to local tryptophan depletion and Kyn accumulation in the tumor, thereby inducing effector T cell dysfunction, promoting regulatory T cell (Treg) proliferation, and ultimately forming a repressive TIME. These factors collectively weaken the survival, proliferation, and sustained anti-tumor capacity of adoptive immune cells in solid tumors, particularly limiting the therapeutic efficacy of CAR-T cell therapy in solid tumors. Therefore, immunometabolic regulation strategies targeting IDO1 are considered an important approach to reshaping the immunosuppressive microenvironment and enhancing the efficacy of cellular immunotherapy.

[0004] NLG919 is a representative small-molecule IDO1 inhibitor that can effectively inhibit the IDO1-mediated tryptophan metabolism pathway, thereby reversing Kyn-dependent immunosuppression and restoring the proliferation and function of effector T cells or CAR-T cells. Studies have shown that NLG919 can enhance the immune response in various tumor models and produce a synergistic anti-tumor effect with immune checkpoint blockade therapy. However, as a small-molecule drug, NLG919 still faces the following problems in practical applications: (1) Systemic administration easily leads to systemic exposure, making it difficult to maintain an effective drug concentration in the tumor; (2) Small-molecule drugs are cleared quickly in vivo, resulting in a limited duration of action; (3) Inhibiting IDO1 alone is difficult to simultaneously solve the problems of immune checkpoint signaling inhibition and insufficient infiltration of adoptive immune cells in solid tumors. In order to improve the tumor delivery efficiency of NLG919 and prolong its duration of action, some studies have attempted to combine it with nanocarriers or other drug delivery systems, but most of these schemes still rely on exogenous carrier materials, which have shortcomings such as complex structures or lack of synergistic immunomodulatory functions. Furthermore, there is currently no existing technology that can integrate NLG919 with immune functional units at the molecular level, while simultaneously possessing self-assembly, delivery, sustained release, and local immunomodulatory capabilities.

[0005] Peptide-drug conjugates, as a novel drug development strategy, can covalently integrate small molecule drugs with functional peptide fragments, thereby improving drug stability and local accumulation capabilities while introducing new biological functions. In particular, self-assembled peptide-drug conjugates can self-assemble into nanofibers in an aqueous environment through non-covalent interactions, and further construct three-dimensional network hydrogels under physiological conditions. These conjugates possess injectability, biocompatibility, and degradability, providing a new technical pathway for the local delivery of drugs or adoptive immune cells.

[0006] However, existing research on peptide hydrogels or peptide-drug conjugates has largely focused on the delivery of chemotherapeutic drugs or vaccine antigens. It has not yet systematically designed small molecules for immunomodulatory functions like NLG919, nor has it integrated immunomodulatory regulation and immune checkpoint blockade into the same molecular structure. Furthermore, it lacks functionalized hydrogel systems capable of efficiently carrying and locally delivering adoptive immune cells (such as CAR-T cells). Therefore, there is an urgent need for a novel peptide-drug conjugate structure centered on NLG919 that maintains IDO1 inhibitory activity while possessing self-assembly and gelation capabilities, and can introduce immunofunctional peptides to synergistically regulate the tumor immune microenvironment. This would provide a new technological solution for the precise local delivery of NLG919 and the application of adoptive immune cell therapy in solid tumors.

[0007] Chinese patent CN118236513A discloses a paclitaxel immunomodulatory hydrogel formulation, its preparation method, and its application, comprising a nanofiber solution and an immunotherapeutic agent added to the nanofiber solution. The paclitaxel immunomodulatory hydrogel formulation can act as a drug reservoir, controlling the slow release of the drug. After a single dose, it activates a long-lasting immune response in vivo, increasing the infiltration of lymphocytes in the tumor microenvironment, thereby effectively inhibiting tumor growth, recurrence, and metastasis.

[0008] Chinese patent CN119454926A discloses a universal hydrogel vaccine based on tumor neoantigen peptides, its preparation method, and its application. The hydrogel vaccine includes a tumor neoantigen peptide hydrogel and an adjuvant loaded on the tumor neoantigen peptide hydrogel. The tumor neoantigen peptide-peptide assembly sequence conjugate assembles in a solvent to form a nanofiber solution, which triggers the formation of a hydrogel under physiological conditions. This method targets different tumor neoantigen peptides and adjusts the amount of valine or phenylalanine in the peptide assembly sequence to achieve the self-assembly of the conjugate in a solvent to form a nanofiber solution. The vaccine adjuvant is then loaded through physical action to obtain a nanofiber solution containing the vaccine adjuvant, thereby achieving personalized cancer immunotherapy.

[0009] While the above-mentioned technical solutions demonstrate the potential of peptide hydrogels in drug sustained release and local delivery, they mainly focus on the delivery of chemotherapeutic drugs or neoantigen peptide vaccines. They are not specifically designed to address the problems of immunosuppressive microenvironment and the insufficient infiltration of adoptive immune cells in solid tumors, nor do they achieve the integration of immune metabolic regulation and immune checkpoint blockade into the same molecular structure to synergistically regulate the tumor immune microenvironment.

[0010] Patent CN115884787A discloses a therapeutic hydrogel material used in conjunction with PD-L1 blocking antibody (aPDL1) modified platelets to deliver CAR-T cells to tumor resection cavities, tissue depressions, or lacunar regions. This patent uses a conventional hydrogel as a physical scaffold to achieve co-enrichment of CAR-T cells and aPDL1-carrying platelets, and utilizes aPDL1-containing platelet-derived microparticles released during postoperative inflammation to enhance local immune blockade. In some embodiments, cytokines are also loaded into nanoparticles and used in conjunction with the hydrogel to maintain the activity and proliferation capacity of CAR-T cells. However, the hydrogel framework of this system is an inert scaffold and does not possess inherent immunomodulatory functions. Immunomodulation mainly relies on the synergistic effect of exogenous platelet-antibody complexes and additional nanocarriers, and does not involve integrating immunomodulatory molecules and immune checkpoint inhibitors into the same molecule to achieve continuous and controllable local immunomodulation through the gel framework itself.

[0011] In summary, the existing technology has not yet formed a comprehensive technical solution that uses NLG919 as the core, constructs an immunomodulatory hydrogel formulation through peptide drug conjugates to achieve continuous immune metabolic regulation, and further applies it to local delivery of CAR-T cells, synergistically improves the tumor immune microenvironment, thereby enhancing the therapeutic effect of CAR-T cell therapy on solid tumors. Summary of the Invention

[0012] The purpose of this invention is to construct a polypeptide-drug conjugate system with the immunomodulatory small molecule NLG919 as its core, enabling it to self-assemble in an aqueous medium and form an immunomodulatory hydrogel scaffold in situ without the need for an exogenous carrier. This scaffold can be used for the local delivery of adoptive immune cells (such as CAR-T cells) and related immunomodulatory agents, thereby synergistically addressing key issues such as IDO1-mediated immunomodulatory inhibition, insufficient infiltration and functional exhaustion of effector cells and adoptive immune cells in solid tumors caused by immune checkpoint signals, on the same platform.

[0013] Based on this, this application provides a method for preparing NLG919-peptide drug conjugates and their hydrogel formulations, as well as their applications.

[0014] This application uses NLG919 as the core drug, and integrates it with self-assembled peptides and immune-functional peptide fragments to form a peptide-drug conjugate. This conjugate can self-assemble into nanofibers in an aqueous environment and form an immunomodulatory hydrogel scaffold under physiological conditions. While providing an injectable local "reservoir", it also has the functions of immune metabolism regulation and immune signal regulation.

[0015] Furthermore, the peptide drug conjugate of this application can be used to efficiently carry and locally deliver adoptive immune cells, represented by CAR-T cells, thereby synergistically improving TIME suppression, adoptive cell infiltration, and effector function in the treatment of solid tumors.

[0016] Hydrogel formulations based on NLG919-peptide drug conjugates represent a novel immunomodulatory hydrogel system that can meet the urgent needs of tumor immunotherapy, particularly for local immune regulation and adoptive immune cell delivery in solid tumors.

[0017] The objective of this invention can be achieved through the following technical solutions: This invention first provides an NLG919-peptide drug conjugate, which is composed of NLG919, a self-assembling peptide, and an immune-functional peptide. The self-assembled polypeptide serves as the backbone, with its N-terminus being a cysteine ​​residue. The self-assembled polypeptide chain has an immune-functional polypeptide linearly linked to its C-terminus. The N-terminus of the self-assembled polypeptide chain is coupled to NLG919 via a disulfide linker through a cysteine ​​side chain thiol group. The NLG919-peptide drug conjugate adopts a tandem structure, forming a multi-segment structure of "NLG919-self-assembled peptide-immune function peptide".

[0018] In one embodiment of the present invention, the immune functional polypeptide is a functional polypeptide capable of regulating the tumor immune microenvironment, selected from one or more of the following classes of polypeptides: Peptides with immune checkpoint regulation effects, including but not limited to CLQKTPKQC, CVRARTR, SNTSESFKFRVTQLAPKAQIKE, NYSKPTDRQYHF, or WGHSHFSHWKGR; Peptides with immune cell co-stimulatory or activating functions, including but not limited to PPRYNLFFLFRFYCSFRRDYLYF, HSFVLFGVNVPFNIIDFQMRVKC, CIEEGQYCFADPYLC or HPFSIKNVFCIWNFFSVY; Peptides with cytokine-like activity, including but not limited to QPWEHVNAIQEAR, DFLLVIP, or KVTAMKCFLL.

[0019] In some embodiments of the present invention, the self-assembled polypeptide may be one or a combination of several of the following: FFY, FFFY, FFYY, K2(SL)6K2, KLDLPVGLIGKLDL, K(SL)6KGPRKLYDY, KFKFEFKFE, VVAA, VVVAAA, VLTKVKTKVPLPTKVEVKVLV, VVAAPLGLAG, GV2Q2HKD, KVKVPPTKVKVKVKVKVKV, RADARADARADA, AGEDQLKHVFS, and K2(QL)6K2.

[0020] In some embodiments of the present invention, the NLG919-peptide drug conjugate may further incorporate a hydrophobic fatty chain. The α-amino group of the N-terminal cysteine ​​residue of the self-assembly peptide is linked to the hydrophobic fatty chain via an amide bond. The hydrophobic fatty chain is used to further promote the self-assembly of the NLG919-peptide drug conjugate. Structurally, the hydrophobic fatty chain and NLG919 are respectively linked to different functional sites of the N-terminal cysteine ​​residue of the self-assembly peptide, thereby jointly participating in the self-assembly behavior of the conjugate.

[0021] In some embodiments of the present invention, the hydrophobic aliphatic chain is selected from one of the alkyl chains corresponding to palmitic acid, lauric acid, octanoic acid, or hexanoic acid.

[0022] More preferably, the immune-functional polypeptide is a PD-L1 antagonistic peptide. D PPA1 or its conserved functional variants. The self-assembled polypeptide is VVAAPLGLAG, with the hydrophobic molecular fatty chain selected from the alkyl chain corresponding to palmitic acid. At this point, the polypeptide-drug conjugate NLG919- D PPA1 is composed of the immunomodulatory molecules NLG919 and PD-L1 antagonist peptides. D It is constructed from PPA1, the self-assembled polypeptide VVAAPLGLAG, and hydrophobic C16 alkyl chains, and can form nanofiber structures in aqueous solution.

[0023] in, D PPA1 refers to a D-type polypeptide. D PPA-1, also written as (D)-PPA-1, CAS No.: 1620813-53-7, has the following structure: The present invention further provides a method for preparing NLG919-peptide drug conjugate, comprising the following steps: S1. Couple NLG919 with a cleavable linker to obtain an NLG919 precursor molecule with a disulfide bond responsive structure; S2. The NLG919 precursor molecule obtained in S1 is conjugated with a self-assembled polypeptide containing an immunomodulatory sequence to obtain the NLG919-peptide drug conjugate.

[0024] In one embodiment of the present invention, step S2 further includes introducing a hydrophobic fatty chain; specifically, the hydrophobic fatty chain is linked to the α-amino group of the N-terminal cysteine ​​of the self-assembled polypeptide via an amide bond; and NLG919 is coupled to the side chain thiol group of the N-terminal cysteine ​​via the cleavable linker to obtain an NLG919-peptide drug conjugate containing a hydrophobic fatty chain.

[0025] In one embodiment of the present invention, the linker is 4-nitrophenyl (3-(pyridin-2-yldithioalkyl)propyl) carbonate. CAS No.: 1610731-08-2, with the following structure: The present invention further provides an immunomodulatory hydrogel formulation, which is made by dissolving NLG919-peptide drug conjugate in an aqueous solvent and allowing it to stand at room temperature to self-assemble into a nanofiber solution.

[0026] The nanofiber solution can be further formed into an immunomodulatory hydrogel with a porous structure under physiological conditions.

[0027] In one embodiment of the present invention, the immunomodulatory hydrogel formulation is loaded with one or more immunotherapeutic agents.

[0028] In some embodiments of the present invention, the immunotherapeutic agent may be one or a combination of several of adoptive immune cells, immune checkpoint inhibitors, or cytokines.

[0029] The adoptive immune cells may be one or more of the following: natural killer cells, lymphokine-activated killer cells, cytokine-induced killer cells, cytotoxic T lymphocytes, chimeric antigen receptor T cells, and chimeric antigen receptor natural killer cells.

[0030] The immune checkpoint blocker may be one or more of the following: PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, TIM-3 antibody, LAG-3 antibody.

[0031] The cytokines may be one or more of the following: IL-15, CXCL-9, CXCL-10, TNF-β, GM-CSF, IL-6, CCL28, CXCL-11, IFN-α, IL-21, IL-2, IFN-β, IL-22, CCL27, IFN-γ.

[0032] The present invention further provides a method for preparing an immunomodulatory hydrogel formulation, comprising the following steps: The NLG919-peptide drug conjugate was dissolved in an aqueous solvent and allowed to stand at room temperature to self-assemble into nanofibers.

[0033] When the immunomodulatory hydrogel formulation is loaded with an immunotherapeutic agent, the method for preparing the immunomodulatory hydrogel formulation loaded with the immunotherapeutic agent includes the following steps: The NLG919-peptide drug conjugate was dissolved in an aqueous solvent and allowed to stand at room temperature to allow it to self-assemble into a nanofiber solution. Immunotherapy agents are added to nanofiber solutions and incubated.

[0034] This method allows for the gentle encapsulation and loading of living cells and bioactive molecules.

[0035] In one embodiment of the present invention, the solvent is selected from either deionized water or phosphate buffer; the solution is allowed to stand at room temperature for 1-3 days.

[0036] In one embodiment of the invention, the assembly process is completed by allowing the mixture to stand at room temperature for 12 to 72 hours.

[0037] In one embodiment of the present invention, the incubation temperature is 37°C and the incubation time is 10 minutes.

[0038] To achieve safe and efficient loading of CAR-T cells and maintain their bioactivity within the gel, for immunomodulatory hydrogel formulations, more preferably, the immunofunctional peptide is a peptide with PD-L1 antagonistic activity. D PPA1 or its conserved functional variants. The self-assembling peptide is VVAAPLGLAG, the hydrophobic molecule is a C16 alkyl chain, and the additional immunotherapeutic agent is the cytokine IL-15. At this point, the in situ immunomodulatory hydrogel formulation for delivering CAR-T cells includes a peptide-drug conjugate NLG919- D PPA1 self-assembles into nanofiber solution in solvent, and the cytokine IL-15 loaded in the nanofiber solution.

[0039] The meanings of the polypeptide sequences in this invention are explained as follows: VVAAPLGLAG refers to a polypeptide with the amino acid sequence VVAAPLGLAG, NYSKPTDRQYHF refers to a polypeptide with the amino acid sequence NYSKPTDRQYHF, and so on. Among them, amino acid A is alanine, D is aspartic acid, E represents glutamic acid, F is phenylalanine, G is glycine, H is histidine, I represents isoleucine, K is lysine, L represents leucine, N is asparagine, P is proline, Q represents glutamine, R is arginine, S is serine, T represents threonine, V is valine, and Y is tyrosine.

[0040] Preferably, the present invention also provides a method for preparing an immunomodulatory hydrogel formulation constructed from the above-mentioned NLG919-peptide drug conjugate, the specific steps of which are as follows: S1. NLG919 is coupled with a cleavable linker to obtain a NLG919 precursor molecule with a disulfide bond-responsive structure. The linker is 4-nitrophenyl (3-(pyridin-2-yldithioalkyl)propyl) carbonate. CAS No.: 1610731-08-2, with the following structure: S2, will include D The N-terminal α-amino group of the self-assembled peptide of PPA1 is linked to a hydrophobic C16 fatty chain via an amide bond to obtain a self-assembled peptide modified with a C16 fatty chain. S3. The NLG919 precursor obtained in S1 is conjugated with the adipose-chain modified self-assembled polypeptide and the immune-functional polypeptide to obtain the polypeptide-drug conjugate NLG919- D PPA1; S4. The polypeptide-drug conjugate NLG919- obtained in S3 DPPA1 was dissolved in a solvent and allowed to stand at room temperature to self-assemble into nanofibers in the solution. Under physiological conditions, a three-dimensional cross-linked structure was further constructed to obtain a porous immunomodulatory hydrogel. S5. Adoptive immune cells such as CAR-T cells and immunomodulators such as cytokines can be added to the nanofiber solution for incubation as needed, to achieve gentle encapsulation and loading of live cells and bioactive molecules.

[0041] In one embodiment of the present invention, the structure of the NLG919 precursor molecule in step S1 is as follows: In one embodiment of the present invention, the dosage relationship of solvent, peptide-drug conjugate, cytokine IL-15, and CAR-T cells is 1 mL: 15-45 mg: 50-150 μg: 1 × 10⁻⁶ μg. 7 -3×10 7 The ratio of aqueous solvent to NLG919-peptide drug conjugate in the hydrogel formulation is 1 mL: 15–45 mg; when the immunotherapy agent is adoptive immune cells and cytokines, the amount of cytokines added is 50–150 μg / 1 mL hydrogel, and the number of adoptive immune cells is 1 × 10⁻⁶. 7 ~3×10 7 One per 1 mL hydrogel.

[0042] This invention further provides the use of the above-mentioned immunomodulatory hydrogel formulation in the preparation of antitumor or anticancer drugs, selected from one of the following uses: Applications in the preparation of antitumor or anticancer drugs; Use in the preparation of formulations for the local delivery of adoptive immune cells.

[0043] The immunomodulatory hydrogel formulation, after being loaded with immunotherapeutic agents (including but not limited to CAR-T cells, other adoptive immune cells, immune checkpoint inhibitors, cytokines, etc.), can be used directly as an antitumor or anticancer drug. When administered, it is injected locally into the tumor lesion or postoperative resection cavity, and can be transformed into a hydrogel state in situ under physiological conditions, forming a local reservoir that combines drugs and cells, which can be used to inhibit tumor growth, reduce the risk of tumor recurrence, and prolong survival.

[0044] In one embodiment of the present invention, when the immunomodulatory hydrogel formulation is used to deliver CAR-T cells, the CAR-T cells can be added to a pre-prepared NLG919- DIn a PPA1 nanofiber solution, after mixing and incubation under mild conditions, a precursor solution for an immunomodulatory hydrogel loaded with CAR-T cells can be obtained. This solution, upon injection into the tumor lesion or postoperative resection cavity, will gel in situ under physiological conditions, forming an in situ immunomodulatory hydrogel loaded with CAR-T cells. Preferably, the incubation temperature is 37°C, and the incubation time is approximately 10 minutes to ensure cell viability and uniform embedding.

[0045] In one embodiment of the present invention, when the immunomodulatory hydrogel formulation is loaded with CAR-T cells, the cell content is preferably 1×10⁻⁶. 7 ~3×10 7 CAR-T cells / mL, meaning that each mL of hydrogel contains 1 × 10⁶ CAR-T cells. 7 ~3×10 7 This allows for the formation of a sufficiently dense population of effector cells locally, thereby achieving an effective anti-tumor effect.

[0046] In situ immunomodulatory hydrogel formulations loaded with CAR-T cells can be used directly as anti-tumor or cancer drugs, and can be converted into a hydrogel state under physiological conditions when used.

[0047] In one embodiment of the present invention, the antitumor or cancer drug is a drug that inhibits tumor growth and recurrence and prolongs the user's survival period.

[0048] In one embodiment of the invention, the antitumor or cancer drug promotes lymphocyte infiltration in the tumor microenvironment, thereby effectively inhibiting tumor growth. CAR-T cells are locally delivered through this hydrogel system and exert a sustained antitumor effect.

[0049] In one embodiment of the present invention, the lymphocytes may be selected from one or more of T cells, B cells, and natural killer cells, preferably genetically engineered CAR-T cells.

[0050] In one embodiment of the present invention, the tumor or cancer may be selected from one or more of the following: glioma, melanoma, endocrine tumor, adenoma, blastoma, desmoidoma, sarcoma, lymphoma, germ cell tumor, rectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, nephroblastoma, lung cancer, colon cancer, breast cancer, gastric cancer, esophageal cancer, cervical cancer, or head and neck cancer.

[0051] The immunomodulatory hydrogel formulation based on peptide-drug conjugates provided by this invention has excellent injectability, allowing direct injection into the lesion area in the form of a nanofiber solution. It rapidly forms a three-dimensional network gel in situ within the body, constructing a local "reservoir" of adoptive immune cells. Unlike traditional hydrogels that rely on exogenous polymeric carriers or non-functional scaffold materials, the hydrogel framework of this invention consists of NLG919 and... D PPA1 is composed of peptide-drug conjugates, and the material itself has both gel-forming ability and immunomodulatory function, enabling the synergistic delivery of immune cells and immunomodulatory molecules without the need for additional carriers.

[0052] The immunomodulatory hydrogel formulation of this invention, when used for CAR-T cell delivery to solid tumors, can effectively improve the infiltration and retention levels of CAR-T cells within the tumor. Simultaneously, NLG919 and... D Sustained release of PPA1 can simultaneously inhibit the IDO1-mediated tryptophan metabolism pathway and block PD-1 / PD-L1 axis-mediated immune checkpoint inhibition locally, thereby regulating the immunosuppressive state in the tumor microenvironment and promoting the expansion of CAR-T cells in the tumor region and the maintenance of effector function.

[0053] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The NLG919-peptide drug conjugate and the immunomodulatory hydrogel formulation constructed by the present invention have the functions of immune metabolism regulation, self-assembly into gel and local cell delivery. It can achieve synergistic sustained release of NLG919 and immune function peptides without the need for additional carriers, and can efficiently carry adoptive immune cells. It solves the multiple limitations of traditional systemic drug delivery and inert scaffold materials in terms of TIME regulation, immune cell infiltration and safety, and has good innovation and application prospects.

[0054] (2) The hydrogel formulation made from NLG919-peptide drug conjugate of the present invention is administered in the form of injectable nanofiber solution. After entering the tumor lesion or postoperative resection cavity, it can form a three-dimensional porous gel in situ, confining the immunomodulatory molecules and adoptive immune cells around the target tissue. This can help reduce the risk of systemic exposure and improve clinical translatability while meeting the local treatment needs.

[0055] (3) The immunomodulatory hydrogel constructed in this invention has good versatility and can be loaded with different types of adoptive immune cells (such as CAR-T cells, CAR-NK cells, etc.) and various cytokines or adjuvants as needed. It is suitable for local administration of various solid tumors and postoperative residual cavities, and expands the application scope of peptide-drug conjugates in the field of tumor immunotherapy. Attached Figure Description

[0056] Figure 1The polypeptide-drug conjugate NLG919- of Example 1 of this invention D The chemical structural formula of PPA1; Figure 2 The polypeptide-drug conjugate NLG919- of Example 1 of this invention D Mass spectrum of PPA1; Figure 3 This is a transmission electron microscope image of NDF in Embodiment 2 of the present invention; Figure 4 This is a diagram of the "solution-gel" transition of NDF in Example 2 of the present invention; Figure 5 This is a diagram showing the drug release characteristics of NDF-Gel in Example 2 of the present invention; Figure 6 This is a scanning electron microscope image of NDF-Gel from Embodiment 2 of the present invention; Figure 7 In Example 3 of this invention, after NDF-Gel treatment, the infiltration of intratumoral Treg cells and endogenous CD8... + T cell proliferation level; Figure 8 This is a three-dimensional confocal image of CAR-T / NDF-Gel in Embodiment 4 of the present invention; Figure 9 This describes the intratumoral release behavior of CAR-T in CAR-T / NDF-Gel in Example 4 of the present invention. Figure 10 This refers to the level of CAR-T cell infiltration and activation within the tumor after CAR-T / NDF-Gel treatment in Example 5 of the present invention. Figure 11 This is a schematic diagram of the use of CAR-T / NDF solution for local filling of the postoperative resection cavity in Embodiment 6 of the present invention; Figure 12 This is an MRI image of a tumor after CAR-T / NDF-Gel treatment in Example 6 of the present invention; Figure 13 This is the survival curve of tumor-bearing mice after CAR-T / NDF-Gel treatment in Example 7 of the present invention. Detailed Implementation

[0057] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, which are intended to explain rather than limit the invention.

[0058] The materials required in the following embodiments are as follows: NLG919 was purchased from Tajimore Chemical Company, USA; all amino acids were purchased from Shanghai Jier Biochemical Co., Ltd.; CAR-T cells were purchased from Zhenjiang Weigen Biotechnology Co., Ltd.; and IL-15 was purchased from InvivoGen, USA. Unless otherwise specified, all other raw materials or processing techniques are commercially available materials or conventional processing techniques in the art.

[0059] The present invention will be further illustrated below with specific embodiments. It should be understood that these embodiments are only used to illustrate the technical solutions of the present invention, and not to limit the present invention.

[0060] Example 1 NLG919-peptide drug conjugate NLG919- D Preparation of PPA1 2,2'-Dithiodipyridine (2 g, 9.08 mmol) and 3-mercapto-1-propanol (1.25 g, 13.62 mmol) were dissolved in 50 mL of methanol and reacted with the solution at room temperature for 6 hours. After the reaction was completed, the product was purified by column chromatography to give the intermediate 3-(2-pyridyldithio)propanol. Subsequently, the obtained 3-(2-pyridyldithio)propanol (1 g, 4.97 mmol) was dissolved together with 4-nitrophenyl chloroformate (1.5 g, 7.45 mmol) and N,N-diisopropylethylamine (DIEA, 0.642 g, 4.97 mmol) in 50 mL of dichloromethane and reacted with the solution at room temperature overnight. The reaction solution was purified by column chromatography to give 4-nitrophenyl (3-(2-pyridyldithio)propyl) carbonate. Next, NLG919 (200 mg, 708.2 μmol), 4-nitrophenyl (3-(2-pyridyldithio)propyl) carbonate (389.26 mg, 1.06 mmol), and 4-dimethylaminopyridine (DMAP, 173.05 mg, 1.43 mmol) were dissolved in 30 mL of dichloromethane, and the mixture was stirred at room temperature for 48 hours under nitrogen protection. The final product, the NLG919 precursor molecule, was purified by column chromatography.

[0061] D PPA1 modified peptide (sequence: N-CVVAAPLGLAG(NYSKPTDRQYHF)) D -C) was prepared by the standard Fmoc solid-state synthesis method. Subsequently, [the following was added]... DPPA1-modified peptides were dissolved in resin (0.25 mmol), palmitic acid (1 mmol), HBTU (peptide coupling reagent, 1 mmol), and DIEA (N,N-diisopropylethylamine, 1 mmol) in 10 mL of dimethylformamide (DMF) and reacted overnight at 55°C with stirring. After the reaction was complete, the product was cleaved from the resin using a TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / water solution (92.5:5:2.5), purified by reversed-phase high-performance liquid chromatography (HPLC), and its molecular weight was verified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOFMS).

[0062] The above C16- D The PPA1-modified peptide and NLG919 precursor molecule were dissolved in 1 mL of dimethyl sulfoxide (DMSO) at a molar ratio of 1:3. The mixture was stirred at room temperature for 36 hours under nitrogen protection to finally obtain the peptide-drug conjugate NLG919- D PPA1. NLG919- D PPA1 is dissolved in deionized water and allowed to stand at room temperature for 24 hours to obtain nanofiber solution NDF.

[0063] Figure 1 The polypeptide-drug conjugate NLG919- in this invention D The chemical structural formula of PPA1 and its mass spectrometry analysis results are as follows: Figure 2 As shown, this confirms NLG919- D Successful synthesis of PPA1.

[0064] Example 2 NLG919- D Preparation and characterization of PPA1 nanofiber NDF and immunomodulatory hydrogel NDF-Gel NLG919- D The preparation method of PPA1 nanofiber solution NDF and immunomodulatory hydrogel NDF-Gel is as follows: The peptide-drug conjugate NLG919- D PPA1 is dissolved in an aqueous solvent (preferably 2 mM) and left to stand at room temperature for 24 h to self-assemble into a nanofiber solution NDF. Then, PBS with a volume of 1 / 10 of the NDF solution is added to the obtained NDF solution and mixed well to trigger the formation of an immunomodulatory hydrogel NDF-Gel.

[0065] Figure 3 The image shown is a transmission electron microscope image of the nanofiber, demonstrating the successful assembly of the nanofiber.

[0066] like Figure 4 The inverted bottle experiment shown demonstrates that NLG919- in this invention... DPPA1 nanofibers (NDF) can transform from a solution state to a gel state (NDF-Gel) under physiological conditions.

[0067] 20 μL of 10×PBS was added to 180 μL of 2 mM NDF solution to form NDF-Gel. The solution was then incubated at 37°C with 1 mL of pH 7.4 PBS. During the 30-day experimental period, 50 μL of supernatant was collected at predetermined time points and replenished with an equal volume of PBS. NLG919- was detected and analyzed using high-performance liquid chromatography (HPLC). D Cumulative release of PPA1.

[0068] like Figure 5 As shown, the hydrogel drug reservoir formed by NDF, NDF-Gel, can achieve continuous release of drug molecules in vitro for up to 30 days, with a cumulative release rate of 74.7%.

[0069] like Figure 6 As shown, the three-dimensional porous gel structure NDF-Gel formed by NDF-triggered nanofibers provides ample physical space for the loading of subsequent immunotherapeutic agents. These immunotherapeutic agents can be CAR-T cells, other adoptive immune cells, cytokines, or immune checkpoint inhibitors, etc., which can be selected and combined by those skilled in the art according to treatment needs.

[0070] Example 3 NDF-Gel continuously regulates the tumor immunosuppressive microenvironment C57BL / 6 mice aged 6-8 weeks were selected, and 2×10⁻⁶ mice were subcutaneously injected into their backs. 6 A B16 melanoma model was established using B16 melanoma cells. On day 10 after modeling, mice were randomly divided into the following groups: (i) PBS treatment group; (ii) NLG919 and... D PPA1 mixed administration (NLG+) D PPA group (NLG919: 6 mg / kg, D (iii) NDF-Gel treatment group (4.24 mM, 100 μL), administered via local injection to the tumor. Subsequently, tumor tissue was collected on day 14 after administration, and single-cell suspensions were obtained by enzymatic digestion. The infiltration level of Treg cells and endogenous CD8+ in the tumor were analyzed by flow cytometry. + T cell proliferation level.

[0071] Figure 7 The study showed the level of (a) Treg cell infiltration and endogenous (b) CD8 infiltration within the tumor 14 days after drug administration. +The results showed that NDF-Gel achieved long-term inhibition of intratumoral Tregs and maintained endogenous CD8 proliferation levels. + The proliferation activity of T cells fully demonstrates the sustained regulatory effect of NDF-Gel on the tumor immune microenvironment.

[0072] Example 4 Immunomodulatory hydrogel NDF-Gel efficiently loads adoptive immune cells and prolongs their retention time in the tumor site (taking CAR-T cells as an example). The prepared NLG919- D PPA1 nanofiber solution NDF contains 1×10 per 1 mL 7 -3×10 7 The CAR-T cells were slowly added to the pre-counted and resuspended CAR-T cell suspension at a certain ratio. After being gently mixed, the mixture was incubated at 37°C for about 10 minutes to allow the CAR-T cells to be evenly dispersed and embedded in the nanofiber network. Then, under physiological conditions, the cells were transformed in situ to form a highly efficient immunomodulatory hydrogel CAR-T / NDF-Gel loaded with CAR-T cells.

[0073] like Figure 8 As shown in the 3D confocal images, NDF-Gel can achieve uniform loading of CAR-T cells.

[0074] Furthermore, a B16 subcutaneous melanoma model was constructed using the method described in Example 3. When the tumor volume reached approximately 200 mm²... 3 At that time, 100 μL of CAR-T / NDF solution pre-incubated as described above (containing 4.24 mM NDF, 2 × 10⁻⁶ mM NDF) was injected locally into the tumor. 6 CAR-T cells and 50 μg IL-15 were injected in situ to form a CAR-T / NDF hydrogel (CAR-T / NDF-Gel). For comparison, free CAR-T cells were injected alone as a control. In vivo fluorescence signals of CAR-T cells were monitored using a small animal in vivo imaging system at days 0, 1, 3, 7, and 15 to assess their retention and distribution within the tumor.

[0075] like Figure 9 As shown, free CAR-T cells were rapidly metabolized and cleared within 5 days after injection, while NDF-Gel significantly prolonged the retention time of CAR-T cells in the tumor, with 22.6% of CAR-T cells still observed on day 15 after administration.

[0076] Example 5 Immunomodulatory hydrogel NDF-Gel enhances the intratumoral infiltration and effector function of CAR-T cells.

[0077] A B16 subcutaneous melanoma model was constructed according to the method described in Example 3. On day 10 after modeling, mice were randomly divided into the following groups: (i) Free CAR-T cell treatment group (2×10⁻⁶ cells / year) 6 (ii) Mixed free drug administration (Bolus) group (NLG919: 6 mg / kg, CAR-T cells: 2 × 10⁻⁶ cells / animal); 6 Individual / each D PPA1: 32.95 mg / kg, IL-15: 50 μg); and (iii) CAR-T / NDF-Gel treatment group (CAR-T cells: 2 × 10⁻⁶). 6 CAR-T cells were injected locally into the tumor (100 μL, IL-15: 50 μg, NDF-Gel: 4.24 mM). Subsequently, tumor tissue was collected on day 3 post-administration, and a single-cell suspension was obtained by enzymatic digestion. CAR-T cell infiltration was analyzed by flow cytometry 3 days after administration.

[0078] Figure 10 The proportion of CAR-T cell infiltration in tumor tissue was shown, and CD4 was further evaluated. + CAR-T cells and CD8 + CAR-T cell activation levels. The results showed that (a) NDF-Gel significantly enhanced the intratumoral infiltration ability of CAR-T cells, and (b, c) simultaneously enhanced their activation levels.

[0079] Example 6 Immunomodulatory hydrogel NDF-Gel delivers CAR-T cells to inhibit postoperative recurrence of glioma. C57BL / 6 mice were fixed in a stereotaxic apparatus, and the skull was exposed by making an incision along the midline of the scalp. A hole was drilled 1 mm to the right of the anterior fontanelle and 2 mm anteriorly, and the needle was inserted perpendicularly 2.5 mm into the hole. Using a microsyringe, 5 μL of a solution containing 1 × 10⁻⁶ mol / L of mol / L was injected. 5 A suspension of GL261 glioma cells was injected into the right striatum region of the brain. After injection, the needle was left in place for 10 minutes, then slowly withdrawn. The burr hole was sealed with bone wax and the incision was sutured, thus establishing an in situ model of the glioma.

[0080] Tumor resection was performed on day 7 after modeling, and CAR-T / NDF solution (CAR-T cells: 2×10⁻⁶) was administered. 6 (Number of animals / animal; NDF-Gel: 42.4mM, 10μL; IL-15: 50μg) was injected into the postoperative resection cavity. Simultaneously, a PBS treatment group and an NDF-Gel treatment group (42.4mM, 10μL; IL-15: 50μg) were set up as control groups.

[0081] like Figure 11As shown, CAR-T / NDF has excellent injectability and can be seamlessly integrated into the cavity filling after glioma resection, forming CAR-T / NDF-Gel in situ.

[0082] Figure 12 The magnetic resonance imaging results showed that mice in both the PBS treatment group and the NDF-Gel treatment group experienced rapid tumor recurrence within 20 days after tumor resection, while the CAR-T / NDF-Gel treatment group significantly inhibited the recurrence of glioma.

[0083] Example 7 Immunomodulatory hydrogel NDF-Gel delivers CAR-T cells, prolonging the survival of tumor-bearing mice.

[0084] A glioma resection model was constructed according to the method in Example 6, and CAR-T / NDF solution (CAR-T cells: 2×10⁻⁶) was used. 6 CAR-T / NDF-Gel (42.4 mM, 10 μL; IL-15: 50 μg) was injected into the postoperative resection cavity. A PBS-treated group and an NDF-Gel-treated group (42.4 mM, 10 μL; IL-15: 50 μg) were set up as control groups. The survival time of mice was monitored and recorded, and survival curves were plotted to evaluate the in vivo therapeutic effect of CAR-T / NDF-Gel.

[0085] Figure 13 The survival curves of mice after CAR-T / NDF-Gel treatment are presented statistically. The results show that before day 23, 5 / 6 of the mice did not experience tumor recurrence; by day 40, all mice were still alive, maintaining a 100% survival rate. Even at the monitoring time of day 60, the CAR-T / NDF-Gel treatment group still maintained a 4 / 6 survival rate, significantly improving survival time compared to the PBS and NDF-Gel treatment groups.

[0086] The above description is merely a preferred embodiment of the present invention, used to illustrate the technical concept of the present invention, and does not constitute a limitation thereof. Although the present invention has been described in detail with reference to specific embodiments, those skilled in the art can still make various modifications, substitutions, or equivalent variations to its technical solutions without departing from the essential spirit of the present invention. Any equivalent changes or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An NLG919-peptide drug conjugate, characterized in that, Composed of NLG919, self-assembling peptides, and immune-functional peptides. The self-assembled polypeptide serves as the backbone, with its N-terminus being a cysteine ​​residue. The C-terminus of the self-assembled polypeptide chain is linked to an immune-functional polypeptide. The N-terminus of the self-assembled polypeptide is coupled to NLG919 via a disulfide linker through a cysteine ​​side chain thiol group. The NLG919-peptide drug conjugate adopts a tandem structure, forming a multi-segment structure of "NLG919-self-assembled peptide-immune function peptide".

2. The NLG919-peptide drug conjugate according to claim 1, characterized in that, The immune-functional polypeptide is a functional polypeptide capable of regulating the tumor immune microenvironment, and is selected from one or more of the following classes of polypeptides: Peptides with immune checkpoint regulation effects, including but not limited to CLQKTPKQC, CVRARTR, SNTSESFKFRVTQLAPKAQIKE, NYSKPTDRQYHF, or WGHSHFSHWKGR; Peptides with immune cell co-stimulatory or activating functions, including but not limited to PPRYNLFFLFRFYCSFRRDYLYF, HSFVLFGVNVPFNIIDFQMRVKC, CIEEGQYCFADPYLC or HPFSIKNVFCIWNFFSVY; Peptides with cytokine-like activity, including but not limited to QPWEHVNAIQEAR, DFLLVIP, or KVTAMKCFLL.

3. The NLG919-peptide drug conjugate according to claim 1, characterized in that, The self-assembled peptides include, but are not limited to, one or more of the following: FFY, FFFY, FFYY, K2(SL)6K2, KLDLPVGLIGKLDL, K(SL)6KGPRKLYDY, KFKFEFKFE, VVAA, VVVAAA, VLTKVKTKVPLPTKVEVKVLV, VVAAPLGLAG, GV2Q2HKD, KVKVPPTKVKVKVKVKVKV, RADARADARADARADA, AGEDQLKHVFS, and K2(QL)6K2.

4. The NLG919-peptide drug conjugate according to claim 1, characterized in that, The NLG919-peptide drug conjugate can also be further incorporating hydrophobic fatty acid chains. The α-amino group of the N-terminal cysteine ​​residue of the self-assembled polypeptide is linked to a hydrophobic fatty chain via an amide bond. This hydrophobic fatty chain further promotes the assembly of the NLG919-peptide drug conjugate. The hydrophobic aliphatic chain is selected from one of the alkyl chains corresponding to palmitic acid, lauric acid, octanoic acid, or hexanoic acid.

5. The NLG919-peptide drug conjugate according to claim 4, characterized in that, The immune-functional polypeptide has PD-L1 antagonistic activity. D PPA1 or its conserved functional variants, the self-assembled polypeptide is VVAAPLGLAG, and the hydrophobic fatty chain is the alkyl chain corresponding to palmitic acid.

6. An immunomodulatory hydrogel formulation, characterized in that, The method involves dissolving the NLG919-peptide drug conjugate as described in any one of claims 1-5 in an aqueous solvent, allowing it to stand at room temperature, and then allowing it to self-assemble into a nanofiber solution. The nanofiber solution can undergo a "solution-gel" transformation under physiological conditions to construct a three-dimensional porous immunomodulatory hydrogel.

7. The immunomodulatory hydrogel formulation according to claim 6, characterized in that, The immunomodulatory hydrogel formulation may be loaded with one or more immunotherapeutic agents.

8. The immunomodulatory hydrogel formulation according to claim 7, characterized in that, The immunotherapy agent is selected from one or a combination of several of adoptive immune cells, immune checkpoint inhibitors, or cytokines. The adoptive immune cells are selected from one or more of the following: natural killer cells, lymphokine-activated killer cells, cytokine-induced killer cells, cytotoxic T lymphocytes, chimeric antigen receptor T cells, and chimeric antigen receptor natural killer cells. 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; The cytokines are selected from one or more of the following: IL-15, CXCL-9, CXCL-10, TNF-β, GM-CSF, IL-6, CCL28, CXCL-11, IFN-α, IL-21, IL-2, IFN-β, IL-22, CCL27, and IFN-γ.

9. The application of the immunomodulatory hydrogel formulation according to any one of claims 6-8, characterized in that, Used to prepare antitumor or anticancer drugs, or to prepare preparations for local delivery of adoptive immune cells.

10. The application of the immunomodulatory hydrogel formulation according to claim 9, characterized in that, The antitumor or cancer drug is one that can promote the infiltration of lymphocytes in the tumor microenvironment, thereby inhibiting tumor growth and recurrence, and prolonging the user's survival period. The lymphocytes are one or more of T cells, B cells, and natural killer cells; The tumor or cancer is selected from one or more of the following: glioma, melanoma, endocrine tumor, adenoma, blastoma, desmoidoma, sarcoma, lymphoma, germ cell tumor, rectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, nephroblastoma, lung cancer, colon cancer, breast cancer, gastric cancer, esophageal cancer, cervical cancer, or head and neck cancer.

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