Cd40l-il-2 fusion protein and its preparation and use in preparing a drug for treating tumors

Abstract

The application discloses a CD40L-IL-2 fusion protein and a preparation method and application thereof in preparing a tumor treatment drug. The fusion protein comprises a CD40L trimer or a variant thereof and a cytokine IL-2 or a variant thereof; the CD40L trimer comprises three CD40L monomers connected. The fusion protein provided by the application can synergistically activate APC-dependent antigen presentation and T cell-mediated immune killing, forms a closed-loop anti-tumor immune response of 'antigen presentation-immune initiation-effect amplification', and provides an effective strategy for treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the CD40L-IL-2 fusion protein, its preparation, and its use in the preparation of drugs for treating tumors. Background Technology

[0002] Cytokine immunotherapy is a therapeutic strategy that activates or enhances anti-tumor immune responses by regulating key signaling molecules in the immune system, such as interleukins and interferons. Cytokines, acting as "messengers" of the immune system, can regulate the function of immune cells such as T cells and NK cells, promote tumor antigen presentation, and reshape the immunosuppressive microenvironment. Interleukin-2 (IL-2) is an important immune cytokine secreted by activated T cells, which can effectively promote the proliferation, differentiation, and survival of effector T cells, memory T cells, and natural killer (NK) cells, thereby enhancing cytotoxic immune responses. IL-2 activates the JAK-STAT and PI3K-AKT signaling pathways by binding to its receptor (IL-2R, composed of α, β, and γ chains), driving the expansion of immune cells and the secretion of anti-tumor molecules such as interferon-γ (IFN-γ). In tumor immunotherapy, IL-2 is used to activate the host immune system (e.g., to treat metastatic melanoma and renal cell carcinoma), but its short half-life and high-dose toxicity (e.g., vascular leakage syndrome) limit its therapeutic efficacy to some extent.

[0003] CD40 ligand (CD40L) is a type II transmembrane protein member of the tumor necrosis factor (TNF) superfamily. CD40L activates downstream immune signaling pathways by binding to CD40 receptors on the surface of dendritic cells and other immune cells, promoting antigen presentation and enhancing T cell function. The role of CD40L is not limited to T cell activation; it can also strengthen tumor-specific immune responses and enhance the body's immune clearance capacity against tumors.

[0004] In recent years, engineered fusion protein technology has provided new ideas for integrating different immune regulatory modules. For example, by optimizing efficacy and safety through domain recombination or Fc fusion, it has shown potential in the design of IL-15 / IL-15Rα and PD-1 / CTLA-4 bispecific antibodies. However, when CD40L is fused with IL, their three-dimensional structures may interfere with each other, leading to steric hindrance or misfolding, affecting solubility and functional activity; receptor binding conflict and signal crosstalk may weaken their respective effects or trigger unintended immunomodulatory effects; fusion proteins have low expression efficiency and are difficult to purify; linkers may introduce immunogenicity, and the difference in half-life between the two can affect pharmacokinetics and targeting; the risk of synergistic immune enhancement or inducing cytokine storm is difficult to predict. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome structural conflicts, functional interference, expression difficulties, and immunogenicity risks while ensuring synergistic effects and avoiding toxicity in the fusion of IL-2 and CD40L. This invention constructs a CD40L-IL-2 fusion protein, overcoming the functional limitations of single cytokines: CD40L drives APC maturation and pro-inflammatory cytokine secretion, promoting naive T cell activation; IL-2 supports the activation and expansion of effector T and NK cells, synergistically activating APC-dependent antigen presentation and T cell-mediated immune killing, thereby forming a closed-loop anti-tumor immune response of "antigen presentation-immune initiation-effector amplification".

[0006] The present invention provides a fusion protein comprising a CD40L trimer or a variant thereof, and the cytokine IL-2 or a variant thereof; the CD40L trimer comprises three linked CD40L monomers.

[0007] In some embodiments, the three CD40L monomers in the CD40L trimer are linked by peptide linkers.

[0008] In some embodiments, the amino acid sequence of the peptide linker is such as (G4S). n As shown, n is an integer from 1 to 5 (n is 1, 2, 3, 4 or 5); n is preferably 2.

[0009] In some embodiments, the amino acid sequence of the peptide linker is shown in SEQ ID NO:5.

[0010] In some embodiments, the amino acid sequence of the CD40L monomer is as shown in SEQ ID NO:3 or 4, or the CD40L monomer is a fragment of a polypeptide with an amino acid sequence as shown in SEQ ID NO:3 or 4.

[0011] In some embodiments, the fusion protein further includes an Fc domain or a variant thereof; the Fc domain is, for example, IgG Fc.

[0012] In some embodiments, the fusion protein further includes a linker; the amino acid sequence of the linker is, for example, (G4S). n As shown, n is an integer from 1 to 5 (n is 1, 2, 3, 4 or 5).

[0013] In some embodiments, the fusion protein provided by the present invention includes at least (i) IL-2 or a variant thereof, (ii) an Fc domain or a variant thereof, and (iii) a CD40L trimer or a variant thereof.

[0014] In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a CD40L trimer, an Fc domain, and the cytokine IL-2, linked sequentially.

[0015] In some implementations, the CD40L trimer is connected to the Fc domain via a first linker, and / or the Fc domain is connected to the cytokine IL-2 via a second linker.

[0016] In some embodiments, the amino acid sequence of the CD40L trimer is shown in SEQ ID NO:2.

[0017] In some implementations, the IgG Fc is derived from mice or humans, and / or the IgG Fc is IgG1 Fc.

[0018] In some embodiments, the IgG Fc is human IgG1 Fc; its amino acid sequence is preferably as shown in SEQ ID NO:9.

[0019] In some embodiments, the amino acid sequence of the cytokine IL-2 is shown in SEQ ID NO:8.

[0020] In some embodiments, the amino acid sequence of the first linker is shown in SEQ ID NO:6.

[0021] In some embodiments, the amino acid sequence of the second linker is shown in SEQ ID NO:7.

[0022] In some specific embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO:1.

[0023] In another aspect, the present invention provides an isolated nucleic acid that encodes the fusion protein provided by the present invention.

[0024] In some embodiments, the sequence of the isolated nucleic acid is shown in SEQ ID NO:11.

[0025] In another aspect, the present invention provides a recombinant vector comprising the isolated nucleic acid provided by the present invention.

[0026] In some implementations, the recombinant vector is a recombinant expression vector or a recombinant cloning vector.

[0027] In some implementations, the recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0028] In some implementation schemes, the prokaryotic expression vector is selected from Escherichia coli expression vectors, Bacillus subtilis expression vectors, and Streptomyces expression vectors; the eukaryotic expression vector is selected from yeast expression vectors, insect expression vectors, and mammalian expression vectors.

[0029] In some embodiments, the mammalian expression vector is pcDNA3.4.

[0030] In another aspect, the present invention provides a transformant comprising the isolated nucleic acid provided by the present invention or the recombinant vector provided by the present invention, wherein the transformant is a non-animal variety and a non-plant variety.

[0031] In some embodiments, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0032] In some embodiments, the eukaryotic cells are yeast cells or mammalian cells, such as CHO-S cells.

[0033] Another aspect of the present invention provides a method for preparing a fusion protein, the method comprising the steps of: culturing the transformant provided by the present invention, and obtaining the fusion protein from the culture.

[0034] In some implementations, the fusion protein is purified from the culture product.

[0035] Another aspect of the present invention provides a pharmaceutical composition comprising the fusion protein provided by the present invention, and a pharmaceutically acceptable carrier.

[0036] In some implementations, pharmaceutically acceptable carriers refer to substances used in pharmaceutical formulations to carry the active pharmaceutical ingredient, which can help improve the drug's stability, solubility, bioavailability, and ease of administration. Common pharmaceutically acceptable carriers include solid carriers, liquid carriers, and semi-solid carriers. Solid carriers include starches (natural polysaccharides, biocompatible and viscous, commonly used as tablet fillers), lactoses (disaccharides, with good flowability, used in oral solid dosage forms), microcrystalline cellulose (the crystalline portion of cellulose, with good compressibility, used as a matrix material for sustained-release tablets), and calcium hydrogen phosphate (a crystalline powder, chemically inert, used as a filler in tablets and capsules). Liquid carriers include water (most commonly used, with good solubility, used in injections and syrups), ethanol (an organic solvent, with good solubility, used in tinctures and medicaments), glycerin (a viscous liquid, with good moisturizing properties, used in syrups and ointments), and polyethylene glycol (a high molecular weight polymer, with good water solubility, used in liquid and semi-solid dosage forms). Semi-solid carriers include petroleum jelly (a petroleum product with good lubricity, used in ointments and eye ointments), lanolin (a natural oil with good water absorption, used in ointments and creams), and beeswax (a natural wax with good plasticity, used in ointments and suppositories). The selection of these carriers needs to be based on a comprehensive consideration of the drug properties, dosage form requirements, and route of administration to ensure the safety, efficacy, and stability of the drug formulation.

[0037] In some embodiments, the pharmaceutical composition further comprises one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0038] Another aspect of the present invention provides the use of the fusion protein, the isolated nucleic acid, the recombinant vector, the transformant, or the pharmaceutical composition provided by the present invention in the preparation of products having any one or more of the following functions:

[0039] 1) Remodeling the tumor microenvironment and immune response;

[0040] 2) Activate the lymph node immune response;

[0041] 3) Activate the function of natural killer cells;

[0042] 4) Promotes the maturation and migration of human dendritic cells;

[0043] 5) Activator CD8 + T cells; and,

[0044] 6) Inhibits tumor cell growth.

[0045] In some implementations, reshaping the tumor microenvironment immune response involves enhancing the activation and function of intratumoral immune cells, such as increasing CD45 levels in tumor tissue. + Activation and function of immune cells.

[0046] In some implementation schemes, activation of the tumor microenvironment immune response promotes CD8 + T cell infiltration and activation.

[0047] In some implementations, CD8 is promoted + T cell infiltration and activation upregulate CD8 in tumor tissue. + The proportion of T cells.

[0048] In some implementations, CD8 is promoted + T cell infiltration and activation enhance intratumoral CD8. + The expression level of CD69, a marker of T cell activation.

[0049] In some implementations, CD8 is promoted + T cell infiltration and activation enhance intratumoral CD8. + T cell effector functions, such as increasing IFN-γ secretion.

[0050] In some implementations, activating the lymph node immune response means enhancing the activation and function of immune cells in the inguinal lymph nodes.

[0051] In some implementations, enhancing the activation and function of inguinal lymph node immune cells specifically promotes CD8. + T cell activation, such as upregulation of CD8 in inguinal lymph nodes + CD69 expression, a marker of T cell activation.

[0052] In some implementations, enhancing the activation and function of inguinal lymph node immune cells is to enhance CD8. + T cell effector function, such as increasing CD8 levels in inguinal lymph nodes + The expression level of IFN-γ, an effector molecule of T cells.

[0053] In some implementations, activating natural killer (NK) cell function involves upregulating the expression of the NK cell activation marker CD69, or enhancing intracellular effector molecules, such as granzyme B (GZMB), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ).

[0054] In some implementations, promoting the maturation of human dendritic cells (DCs) is to enhance the activation and maturation of human DCs, for example by upregulating the expression levels of key activation / maturation markers on the surface of human DCs, such as CD80, CD86, CD40, or CD83.

[0055] In some implementation schemes, human CD8 is activated. + T cells promote activation and proliferation; for example, by increasing CD8. + The expression of CD69, a marker of T cell activation, and / or the expression of Ki-67, an indicator of enhanced intracellular proliferation.

[0056] In some implementation schemes, human CD8 is activated. + T cells enhance their killing function, for example, by increasing CD8+. + Expression of functional effector molecules of T cells, namely CD8 + Functional effector molecules of T cells include, for example, granzyme B (GZMB), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ).

[0057] In some implementations, the tumor cells are solid tumors, such as melanoma cells.

[0058] In another aspect, the present invention provides a kit comprising the fusion protein provided by the present invention or the pharmaceutical composition provided by the present invention.

[0059] In some embodiments, the kit further includes (i) an apparatus for administering the fusion protein or the pharmaceutical composition, and / or (ii) instructions for use.

[0060] Another aspect of the present invention provides a pillbox set, the pillbox set comprising pillbox A and pillbox B, wherein:

[0061] The medicine box A contains the fusion protein provided by the present invention or the pharmaceutical composition provided by the present invention.

[0062] In some embodiments, the kit B contains other fusion proteins for treating solid tumors or pharmaceutical compositions containing said other fusion proteins, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0063] In some implementations, the tumor is a solid tumor, such as melanoma.

[0064] Another aspect of the present invention provides a co-stimulation system comprising the fusion protein provided by the present invention or the isolated nucleic acid provided by the present invention.

[0065] In some implementations, the co-stimulation system further comprises an adjuvant.

[0066] In another aspect, the present invention provides a vaccine comprising the fusion protein provided by the present invention, the isolated nucleic acid provided by the present invention, or the co-stimulation system provided by the present invention.

[0067] Another aspect of the present invention provides a method for promoting the activation and maturation of dendritic cells, the method comprising contacting the dendritic cells with the fusion protein provided by the present invention, the pharmaceutical composition provided by the present invention, the co-stimulation system provided by the present invention, or the vaccine provided by the present invention.

[0068] In some implementations, the method is for non-diagnostic and non-therapeutic purposes.

[0069] Another aspect of the present invention provides a method to promote CD8 + A method for T cell proliferation and activation, the method comprising causing CD8 + T cells come into contact with the fusion protein, pharmaceutical composition, co-stimulation system, or vaccine provided by the present invention.

[0070] In some implementations, the method is for non-diagnostic and non-therapeutic purposes.

[0071] Another aspect of the present invention provides a method for enhancing NK cell proliferation and activation, the method comprising contacting the NK cells with the fusion protein provided by the present invention, the pharmaceutical composition provided by the present invention, the co-stimulation system provided by the present invention, or the vaccine provided by the present invention.

[0072] In some implementations, the method is for non-diagnostic and non-therapeutic purposes.

[0073] Another aspect of the present invention provides a method for inhibiting tumor cell growth, the method comprising contacting the tumor cells with the fusion protein provided by the present invention, the pharmaceutical composition provided by the present invention, the co-stimulation system provided by the present invention, or the vaccine provided by the present invention.

[0074] In some implementations, the method is for diagnostic and therapeutic purposes.

[0075] In some implementations, the tumor cells are solid tumor cells, such as melanoma cells.

[0076] Another aspect of the present invention provides a method for creating a pro-inflammatory environment in tissue surrounding diseased cells, the method comprising contacting the tissue with an effective amount of the fusion protein provided by the present invention, the pharmaceutical composition provided by the present invention, the co-stimulation system provided by the present invention, or the vaccine provided by the present invention.

[0077] Another aspect of the present invention provides a method for treating tumors, the method comprising administering to a subject in need a therapeutically effective amount of the fusion protein provided by the present invention, the pharmaceutical composition provided by the present invention, the co-stimulation system provided by the present invention, or the vaccine provided by the present invention, or administering to a subject in need a kit or a pharmacopoeia provided by the present invention with the active ingredient reaching a therapeutically effective amount.

[0078] In this invention, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0079] In this invention, the term "active ingredient" refers to the effective ingredient with therapeutic effect in the kit or pharmaceutical kit, namely the fusion protein or pharmaceutical composition described in this invention. When referring to "the active ingredient reaching a therapeutically effective amount," it means that the fusion protein or pharmaceutical composition can achieve a blood or tissue concentration sufficient to produce the expected therapeutic effect in the patient after administration.

[0080] In some implementations, the tumor is a solid tumor, such as melanoma.

[0081] Another aspect of the present invention provides the fusion protein, the pharmaceutical composition, the kit, the pharmaceutical kit, the co-stimulation system, or the vaccine provided by the present invention for the treatment of tumors.

[0082] In some implementations, the tumor is a solid tumor, such as melanoma.

[0083] Another aspect of the present invention provides a method for increasing the response of a subject to an antigen, the method comprising administering to the subject an effective amount of the fusion protein provided by the present invention, the pharmaceutical composition, the kit, the cassette, the co-stimulation system, or the vaccine.

[0084] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0085] The reagents and raw materials used in this invention are all commercially available.

[0086] The positive and progressive effects of this invention are as follows:

[0087] The CD40L-IL-2 fusion protein constructed in this invention exhibits superior antitumor effects compared to the combined drug administration group (IL-2+CD40L group). It can activate the immune response in the tumor microenvironment, enhance tumor immune infiltration, and thus exert an antitumor effect. Furthermore, it can increase the proportion of dendritic cells (DCs) in inguinal lymph nodes and enhance their recruitment and activation of CD8+ cells. + The fusion protein exhibits the ability to activate T / NK cells, thus demonstrating a dual activation effect and further enhancing its anti-tumor activity; it effectively activates dendritic cells, natural killer cells, and CD8+. + The ability of T cells is superior to that of single-drug groups with corresponding functions. Attached Figure Description

[0088] Figure 1A This is a schematic diagram of the fusion protein structure.

[0089] Figure 1B This is an SDS-PAGE gel image of the fusion protein in both reduced and non-reduced states.

[0090] Figure 1C This is the HPLC analysis chromatogram of the fusion protein.

[0091] Figure 2 This demonstrates the in vivo antitumor activity of the fusion protein.

[0092] Figure 3AThe percentage of CD45+ white blood cells in mouse melanoma tissue after treatment with the fusion protein.

[0093] Figure 3B CD8 in mouse tumor tissue after treatment with the fusion protein + The proportion of T cells.

[0094] Figure 3C CD8 in mouse tumor tissue after treatment with the fusion protein + The expression level of CD69, a marker of T cell activation.

[0095] Figure 3D CD8 in mouse tumor tissue after treatment with the fusion protein + The amount of IFN-γ secreted by T cell effector molecules.

[0096] Figure 4 The fusion protein enhances the activation and function of dendritic cells in mouse inguinal lymph nodes.

[0097] Figure 5A CD8 in inguinal lymph nodes after fusion protein treatment + The expression level of CD69, a marker of T cell activation.

[0098] Figure 5B CD8 in inguinal lymph nodes after fusion protein treatment + The amount of interferon-γ (IFN-γ), an effector molecule of T cells, secreted.

[0099] Figure 6 The fusion protein significantly upregulated the expression level of CD69, an activation marker of natural killer cells (NK cells).

[0100] Figure 7 The fusion protein significantly upregulated the expression levels of DC surface activation and maturation markers (including CD80, CD86, CD40, and CD83) and migration-related receptor CCR7.

[0101] Figure 8A Treatment of the fusion protein significantly increased CD8 + Expression of CD69, a marker of T cell activation.

[0102] Figure 8B The fusion protein enhanced the level of the intracellular proliferation marker (Ki-67).

[0103] Figure 8C CD8 was enhanced for the fusion protein + The level of granzyme B (GZMB), a functional effector molecule of T cells.

[0104] Figure 8D CD8 was enhanced for the fusion protein+ The level of tumor necrosis factor-α (TNF-α), a functional effector molecule of T cells.

[0105] Figure 8E CD8 was enhanced for the fusion protein + The level of interferon-γ (IFN-γ), a functional effector molecule of T cells.

[0106] Figure 9A The fusion protein significantly enhanced the expression of CD69, an activation marker of NK cells.

[0107] Figure 9B The fusion protein significantly enhanced the expression of intracellular effector molecules (GZMB) in NK cells.

[0108] Figure 9C The fusion protein significantly enhanced the expression of intracellular effector molecules (TNF-α) in NK cells.

[0109] Figure 9D The fusion protein significantly enhanced the expression of intracellular effector molecules (IFN-γ) in NK cells. Detailed Implementation

[0110] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0111] Example 1: Expression and purification of fusion protein

[0112] This embodiment provides a CD40L-IL-2 fusion protein (CD40L-Fc-IL-2), the structural diagram of which is shown below. Figure 1A As shown, from the N-terminus to the C-terminus, it includes a CD40L trimer, a first linker, an Fc domain, a second linker, and IL-2. The CD40L trimer consists of three CD40L monomers covalently linked together by a peptide linker, and its amino acid sequence is as follows:

[0113] The amino acid sequence of the CD40L-IL-2 fusion protein (SEQ ID NO:1):

[0114] MGWSCIILFLVATATGVHSNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGGGGSGGGSQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGGGGSGGGSQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGGGGSGGGGSGGGGSASEPKSCDKTHTCPPCPAPELLGDDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0115] Amino acid sequence of CD40L trimer (SEQ ID NO:2):

[0116] NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGGGGSGGGSQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGGGGSGGGSQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

[0117] Amino acid sequence of CD40L monomer (SEQ ID NO: 3):

[0118] NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

[0119] Amino acid sequence of CD40L monomer (SEQ ID NO: 4):

[0120] QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

[0121] Peptide linker: GGGGSGGGS (SEQ ID NO: 5)

[0122] First linker: GGGGSGGGGSGGGGS (SEQ ID NO: 6)

[0123] Second connector: GGGGS (SEQ ID NO:7)

[0124] The amino acid sequence of IL-2 (SEQ ID NO:8)

[0125] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK PLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0126] The amino acid sequence of the Fc domain (SEQ ID NO:9)

[0127] ASEPKSCDKTHTCPPCPAPELLGDDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP RPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0128] This fusion protein was prepared through the following steps:

[0129] Plasmid preparation: Based on the amino acid sequence and signal peptide sequence (MGWSCIILFLVATATGVHS, SEQ ID NO:10) of the CD40L-IL-2 fusion protein, the target DNA sequence was designed, optimized and synthesized, and the complete sequence was subcloned into the pcDNA3.4 vector to prepare transfection-grade plasmids on a large scale.

[0130] Target DNA sequence (SEQ ID NO:11):

[0131]

[0132] Transfected cells: One day before transfection, CHO-S cells were passaged and seeded at a density of 3 × 10⁶ cells / year. 6 -4×10 6 Cells were allowed to grow overnight at a concentration of [number] cells / mL. On the day of transfection, viable cell density and survival rate were measured, with a cell density reaching approximately 7 × 10⁶ cells / mL. 6 -1×10 7 The cell density was 95-99% (cells / mL). The cells were diluted to a final density of 6 × 10⁶ cells / mL using fresh, pre-warmed Expi CHO medium. 6 Cells / mL. Before transfection, calculate the plasmid volume at 1.0 μg / mL (transfection volume). The volume of the transfection reagent ExpiFectamine CHO is 4 times the plasmid volume. Dilute the plasmid and transfection reagent with OptiPRO medium at a volume ratio of 1:12.5, and gently invert to mix 3 times. Add the diluted ExpiFectamine CHO reagent to the diluted DNA, and invert to mix 3 times. Incubate the ExpiFectamine CHO / plasmid DNA complex at room temperature for 1-5 minutes, then slowly add the solution to a shake flask containing the cells to be transfected, gently rotating the flask during the addition. Then place the flask in a CO2 incubator with shaking at 37°C, 8% CO2, and 80 rpm. On days 1 and 5 after transfection, add 0.12% ExpiCHO enhancer and 5% ExpiCHO feeder by culture volume. Harvest the cell culture supernatant after 10 days for purification.

[0133] Purification and Analysis: The cell culture medium was centrifuged and then filtered. The filtered cell culture supernatant was loaded into MabSelect SuRe. TM LX was operated at the appropriate flow rate. After washing and eluting with 50 mM citrate (pH 3.0), the eluted fraction was mixed and exchanged with buffer to the final formulation buffer.

[0134] The purified protein was subjected to SDS-PAGE ( Figure 1B ), HPLC Figure 1C The molecular weight and purity were determined by analysis. Figure 1A This is a schematic diagram of the structure of the fusion protein.

[0135] Example 2: The fusion protein exhibits antitumor activity in vivo.

[0136] This study aimed to investigate the in vivo antitumor activity of a fusion protein by evaluating its inhibitory effect on the growth of mouse melanoma B16 (ATCC). The experimental procedures and results are detailed below: Six- to eight-week-old male C57 / BL6N mice (purchased from Spiford Biotechnology Co., Ltd.) were used. 3 × 10^5 B16F10 cells / 100 μL were injected subcutaneously into the right axilla of each mouse. The mice were then randomly divided into five groups (n=5 per group): a blank control group (Ctrl group), a single-drug group (CD40L-FC group), a single-drug group (IL-2-FC group), a combination drug group (IL-2+CD40L group), and a fusion protein group (CD40L-IL-2 group). Seven days after subcutaneous tumor construction, the corresponding protein or blank control (PBS) was injected via tail vein every two days at a dose of 10 μg (PBS solvent) for a total of four injections. Tumor volume was measured during each injection.

[0137] As in this embodiment Figure 2 The results showed that the tumor volume in the fusion protein group (CD40L-IL-2 group) was significantly smaller than that in the single-drug groups (CD40L-FC group and IL-2-FC group), indicating that the fusion protein has significant anti-tumor activity. Meanwhile, the tumor volume in the fusion protein group (CD40L-IL-2 group) was slightly smaller than that in the combination therapy group (IL-2+CD40L group), suggesting that the anti-tumor effect was slightly better than the combination therapy.

[0138] Example 3: Fusion protein enhances the activation and function of immune cells in mouse tumors.

[0139] This study aimed to investigate the regulatory role of fusion proteins in the tumor immune microenvironment of mouse melanoma B16 (ATCC) and their immunomodulatory function in vivo. Tumor tissue samples were collected from tumor-bearing mice (Example 2), and single-cell suspensions were prepared using a combination of enzymatic digestion and mechanical grinding. The cell suspensions were then washed 1-2 times with PBS in 1.5 mL centrifuge tubes, and the tumor cells were resuspended in T-cell culture medium for cell counting. The cell density was adjusted to 1 × 10⁻⁶ cells / mL. 6Cells were seeded at 1 mL / well in 24-well plates. In the experimental group, 1×T cell stimulant (containing a leukocyte activation mixture and the protein transport inhibitor Brefeldin A (BD Biosciences)) was added, and cells were stimulated for 6 hours at 37°C and 5% CO2. After cell collection, cells were centrifuged at 300g for 5 minutes at 4°C and washed once with pre-chilled FACS buffer (PBS + 2% FBS + 0.1% NaN3). Surface staining was then performed: anti-mouse CD45, CD3, CD8, and CD69 antibodies (Biolegend) were added, and cells were incubated on ice in the dark for 30 minutes, followed by washing twice with FACS buffer. Intracellular staining was then performed: Cell fixation and permeabilization were performed at room temperature (using Invitrogen eBioscienceFoxp3 / transcription factor flow cytometry fixation and permeabilization buffer). After fixation for 30 minutes, the cells were washed with permeabilization buffer. Cells were resuspended and incubated with anti-IFN-γ antibody (Biolegend) for 30 minutes at room temperature in the dark, followed by two more washes with permeabilization buffer. Finally, cells were resuspended in 500 μL of FACS buffer and filtered through a 40 μm filter. Flow cytometry analysis was performed, and after compensation calibration, ≥1,000 CD45 cells were collected from each sample. + Cells, and then analyzed CD45 infiltrating tumors. + Cells and CD8 + T cell ratio and CD8 + CD69 in T cells + Activation rate and the proportion of IFN-γ cytokine secretion.

[0140] As in this embodiment Figures 3A-3D As shown, after treatment with the CD40L-IL-2 fusion protein, CD45 in mouse melanoma tissue decreased. + The proportion of white blood cells increased significantly ( Figure 3A This indicates that the fusion protein can effectively promote the infiltration of immune cells into the tumor site. More specifically, CD8+ in the tumor microenvironment... + The proportion of T cells increased significantly. Figure 3B ), and the expression level of its activation marker CD69 ( Figure 3C ) and the amount of secretion of the effector molecule IFN-γ ( Figure 3D Both were significantly enhanced, confirming that the CD40L-IL-2 fusion protein can not only promote CD8 + T cell recruitment to tumor tissue can also effectively activate tumor-infiltrating CD8 cells. + T cells activate and kill cells, thus exerting a significant anti-tumor effect.

[0141] Example 4: Fusion protein enhances the activation and function of immune cells in mouse inguinal lymph nodes

[0142] Lymph nodes, as secondary lymphoid organs, are key sites for immune cell interactions and the initiation of adaptive immune responses. In this microenvironment, dendritic cells (DCs), as professional antigen-presenting cells, activate T cells and NK cells through the MHC molecule-co-stimulatory signaling axis, thereby forming a complex immune regulatory network. Studies have shown that bidirectional communication and dynamic balance between DCs and T / NK cells are crucial for effective anti-tumor immunity: (1) DCs as the central nervous system: integrate antigen information and activate T / NK cells through MHC and co-stimulatory molecules. (2) Bidirectional communication: NK cells regulate DC function through cytokine feedback; T cell polarization affects NK activity. (3) Dynamic balance: positive / negative feedback loops ensure effective defense while avoiding excessive inflammation. The analysis of these interactions provides key targets for the design of fusion protein drugs. The goal of this experiment is to explore the effect of fusion proteins in synergistically enhancing anti-tumor immune responses in vivo.

[0143] In this experimental example, inguinal lymph nodes from tumor-bearing mice in Example 2 were selected and placed in pre-chilled RPMI 1640 medium containing 2% fetal bovine serum (FBS). The cells were mechanically ground through a 70 μm sieve (using a syringe plunger for gentle pressure) to prepare a cell suspension, followed by centrifugation at 300g (4°C, 5 minutes). 1 mL of erythrocyte lysis buffer (BD Pharm Lysate) was added. TM Lyse the cells at room temperature for 5 minutes, then wash twice with PBS. Resuspend the T cells in culture medium and adjust the cell density to 1×10⁻⁶. 6 Cells were seeded at 1 mL / well in 24-well plates, and 1×T cell stimulator (containing a leukocyte activation mixture and the protein transport inhibitor Brefeldin A (BD Biosciences)) was added. Cells were cultured at 37°C and 5% CO2 for 4 hours for stimulation. After stimulation, cells were collected by centrifugation and subjected to the following steps: ① Surface staining: Incubation on ice for 30 minutes in the dark using anti-mouse CD45, CD3, NK1.1, CD8, and CD69 antibodies (BioLegend); ② Intracellular staining: Cell fixation and permeabilization were performed at room temperature (using Invitrogen eBioscience Foxp3 / transcription factor flow cytometry fixation and permeabilization buffer). After fixation for 30 minutes, cells were washed with permeabilization buffer. Cells were resuspended and incubated with anti-IFN-γ antibody (Biolegend) at room temperature in the dark for 30 minutes, and finally washed twice with permeabilization buffer. Cells were then resuspended in FACS buffer and filtered. ≥10,000 CD45 cells were collected by flow cytometry. + Cells, and then DC / CD8 were analyzed. + The immune activation state of T / NK cells.

[0144] As in this embodiment Figure 4-6 As shown, after treatment with the CD40L-IL-2 fusion protein, typical dendritic cells (CD11c) in the inguinal lymph nodes of experimental mice... + HLA-DR + The proportion of [a specific cell type] was significantly increased, and the expression levels of its surface activation markers CD80, CD86, and CD40 were significantly upregulated. Compared with the single-drug control groups (CD40L-Fc group and IL-2-Fc group), the CD40L-IL-2 fusion protein significantly enhanced the activation level of dendritic cells in inguinal lymph nodes. Further experimental results showed that the CD40L-IL-2 fusion protein also promoted the activation of CD80, CD86, and CD40 in inguinal lymph nodes. + Upregulation of the expression of CD69, a marker of T cell activation, and interferon-γ (IFN-γ), an effector molecule. Figure 5A , Figure 5B Furthermore, CD69, a marker of natural killer cell (NK cell) activation, also showed a significant increasing trend. Figure 6 This indicates that the fusion protein may enhance the cytotoxic function of NK cells through synergistic effects. Based on the above experimental results, the CD40L-IL-2 fusion protein significantly enhances the immune response of inguinal lymph nodes through a dual mechanism of action: (1) upregulating the proportion of dendritic cells and promoting their activation and maturation; (2) enhancing the resistance of dendritic cells to CD8+. + The ability to recruit and activate T cells and NK cells can synergistically enhance the anti-tumor immune response.

[0145] The experimental data provided in this example demonstrate that the CD40L-IL-2 fusion protein can effectively activate and coordinate the functions of various immune cells, and has important application prospects in anti-tumor immunotherapy.

[0146] Example 5: Fusion protein enhances the activation and function of human immune cells

[0147] Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy volunteers using Ficoll density gradient centrifugation, and three sets of experiments were performed simultaneously. First, dendritic cell (DC) maturation induction experiments were conducted: PBMCs were isolated using the adherent method and cultured for 5 days in RPMI 1640 medium containing 500 U / mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) (conditions: 37°C, 5% CO2, half-medium change every 48 hours) to obtain immature DCs. Subsequently, the cells were centrifuged at 3 × 10⁻⁶ cells / mL. 5Cells were added to wells (24-well plates, 500 μL) at densities of 500 ng / mL CD40L-IL-2 fusion protein or phosphate-buffered saline (PBS) for 24 hours. After treatment, cells were pretreated with FcX inhibitor (BioLegend), and the mean fluorescence intensity (MFI) of CD80, CD86, and CD83 surface markers was measured. The experiment was repeated three times, with each experiment performed three independent technical replicates. Natural killer (NK) cells and CD83 cells were then analyzed. + T cell functional response assay: PBMCs were administered at a concentration of 3 × 10⁻⁶ 5 Cells were seeded at a density per well in 96-well plates and treated with the same dose for 24 hours. Following treatment, surface staining (NK cells: CD3 / CD56 / CD69; CD8) was performed simultaneously. + T cells (CD3 / CD8 / CD69) and intracellular staining (using GZMB, Ki-67, TNF-α, and IFN-γ antibodies (BioLegend), reacted at 4°C in the dark for 45 minutes after membrane perforation). Flow cytometry was used to analyze CD3-CD56. + NK cell population and CD3 + CD8 + The positivity rates of various indicators in the T cell population were measured, and the antibodies used were all purchased from BioLegend.

[0148] As in this embodiment Figure 7 As shown in Figure 9, dendritic cells (DCs) treated with CD40L-IL-2 fusion protein and CD8+ + The expression levels of activation and function-related markers of T cells and natural killer cells (NK cells) were significantly increased. Compared with the single-drug control group, the CD40L-IL-2 fusion protein synergistically enhanced the activation and function of the above-mentioned immune cells.

[0149] Specifically, such as Figure 7 As shown, the CD40L-IL-2 fusion protein significantly upregulated the expression levels of DC surface activation and maturation markers (including CD80, CD86, CD40, and CD83) and migration-related receptor CCR7, indicating that the fusion protein can effectively promote the in vitro activation, maturation, and migration of DCs.

[0150] like Figures 8A-8E As shown, treatment with the CD40L-IL-2 fusion protein significantly increased CD8 + CD69, a marker of T cell activation. Figure 8A The expression of ) was enhanced, while the intracellular proliferation marker (Ki-67, Figure 8B ) and functional effector molecules (including granzyme B (GZMB, Figure 8C ), tumor necrosis factor-α (TNF-α, Figure 8D ) and interferon-γ (IFN-γ, Figure 8E The level of CD8+ demonstrates that the fusion protein can effectively enhance CD8+. + T cell activation, proliferation, and cytotoxicity.

[0151] In addition, such as Figure 9A As shown in Figure D, the CD40L-IL-2 fusion protein significantly increased the expression of CD69, an activation marker of NK cells, and intracellular effector molecules (GZMB, TNF-α, IFN-γ), indicating that the fusion protein can efficiently activate NK cells and enhance their killing function.

[0152] In summary, the CD40L-IL-2 fusion protein exhibits activity against human DCs and CD8+ in in vitro experiments. + The multiple synergistic activation effects of T cells and NK cells have significant immunomodulatory potential.

Claims

1. A fusion protein, characterized in that, The fusion protein is a CD40L trimer, an Fc domain, and the cytokine IL-2 connected sequentially from the N-terminus to the C-terminus; the CD40L trimer is three CD40L monomers connected by a peptide linker. The CD40L trimer is connected to the Fc domain via a first linker, and the Fc domain is connected to the cytokine IL-2 via a second linker; the amino acid sequences of the peptide linker, the first linker, and the second linker are shown as (G4S)n, where n is an integer from 1 to 5; The amino acid sequence of the CD40L trimer is shown in SEQ ID NO:

2.

2. The fusion protein as described in claim 1, characterized in that, The fusion protein satisfies one or more of the following: 1) The amino acid sequence of the peptide linker is shown in SEQ ID NO: 5; 2) The amino acid sequence of the CD40L monomer is as shown in SEQ ID NO: 3 or 4, or the CD40L monomer is a fragment of a polypeptide with an amino acid sequence as shown in SEQ ID NO: 3 or 4; and, 3) The Fc domain is IgG Fc.

3. The fusion protein as described in claim 2, characterized in that, The fusion protein satisfies one or more of the following: 1) The IgG Fc is derived from mice or humans, and / or the IgG Fc is IgG1 Fc; 2) The amino acid sequence of the cytokine IL-2 is shown in SEQ ID NO: 8; 3) The amino acid sequence of the first linker is shown in SEQ ID NO: 6; and, 4) The amino acid sequence of the second linker is shown in SEQ ID NO:

7.

4. The fusion protein as described in claim 3, characterized in that, The IgG1 Fc is human IgG1 Fc.

5. The fusion protein as described in claim 4, characterized in that, The amino acid sequence of the human IgG1 Fc is shown in SEQ ID NO:

9.

6. The fusion protein as described in claim 5, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

1.

7. An isolated nucleic acid, characterized in that, The isolated nucleic acid encodes the fusion protein as described in any one of claims 1-6.

8. The isolated nucleic acid as described in claim 7, characterized in that, The sequence of the isolated nucleic acid is shown in SEQ ID NO:

11.

9. A recombinant vector, characterized in that, The recombinant vector contains the isolated nucleic acid as described in claim 7 or 8.

10. The recombinant vector as described in claim 9, characterized in that, The recombinant vector is a recombinant expression vector or a recombinant cloning vector.

11. The recombinant vector as described in claim 10, characterized in that, The recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

12. The recombinant vector as described in claim 11, characterized in that, The prokaryotic expression vector is selected from Escherichia coli expression vector, Bacillus subtilis expression vector and Streptomyces expression vector; the eukaryotic expression vector is selected from yeast expression vector, insect expression vector and mammalian expression vector.

13. The recombinant vector as described in claim 12, characterized in that, The mammalian expression vector is pcDNA3.

4.

14. A transformant, characterized in that, The transformant comprises the isolated nucleic acid as described in claim 7 or 8 or the recombinant vector as described in any one of claims 9-13, wherein the transformant is a non-animal variety and a non-plant variety.

15. The transformant as described in claim 14, characterized in that, The host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

16. The transformant as described in claim 15, characterized in that, The eukaryotic cells are yeast cells or mammalian cells.

17. The transformant as described in claim 16, characterized in that, The mammalian cells in question are CHO-S cells.

18. A method for preparing a fusion protein, characterized in that, The method comprises the following steps: culturing the transformant as described in any one of claims 14-17, and obtaining the fusion protein from the culture.

19. The method as described in claim 18, characterized in that, The fusion protein was obtained through purification.

20. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the fusion protein as described in any one of claims 1-6, and a pharmaceutically acceptable carrier.

21. The use of the fusion protein of any one of claims 1-6, the isolated nucleic acid of any one of claims 7 or 8, the recombinant vector of any one of claims 9-13, the transformant of any one of claims 14-17, or the pharmaceutical composition of any one of claims 20 in the preparation of a product that inhibits the growth of melanoma cells.

22. A reagent kit, characterized in that, The kit contains the fusion protein as described in any one of claims 1-6 or the pharmaceutical composition as described in claim 20.

23. The kit according to claim 22, characterized in that, The kit also includes (i) an apparatus for administering the fusion protein or the pharmaceutical composition, and / or (ii) instructions for use.

24. A medicine box set, characterized in that, The pillbox set includes pillbox A and pillbox B, wherein: The cassette A contains the fusion protein as described in any one of claims 1-6 or the pharmaceutical composition as described in claim 20.

25. A co-stimulation system, characterized in that, The co-stimulation system comprises the fusion protein as described in any one of claims 1-6.

26. The co-stimulation system as described in claim 25, characterized in that, The co-stimulation system also includes an adjuvant.

27. A vaccine, characterized in that, The vaccine comprises a fusion protein as described in any one of claims 1-6 or a co-stimulatory system as described in claim 25 or 26.

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