Cd40l-il-21 fusion protein and its preparation and use in preparing a drug for treating tumors

Abstract

The application discloses a CD40L-IL-21 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-21 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-21 fusion protein, its preparation, and its use in the preparation of drugs for treating tumors. Background Technology

[0002] Tumor immunotherapy is a biological treatment method that activates or enhances the body's own immune system to recognize and attack tumor cells, bringing breakthroughs to cancer treatment. However, its efficacy is significantly limited in "cold tumors" (tumors with low immune infiltration). Cold tumors are characterized by an immunosuppressive microenvironment, low tumor mutational burden, and lack of T cell infiltration, resulting in a response rate of less than 20% for immune checkpoint inhibitors or CAR-T therapy. Current strategies for cold tumors focus on "immunotransformation," promoting T cell activation, reprogramming the tumor microenvironment, facilitating T cell transport, and enabling T cells to infiltrate the tumor more effectively, helping "cold tumors" transform into "hot tumors" and improving the efficiency of immunotherapy.

[0003] Cytokine immunotherapy has shown unique potential in the treatment of cold tumors. By reshaping the immunosuppressive microenvironment and activating the immune response in cold tumors, it has become an important strategy for overcoming resistance to immune checkpoint inhibitors. For example, the anti-PD-L1 / IL-15 prodrug (LH05) conjugates IL-15 with an anti-PD-L1 antibody, allowing IL-15 to promote CD8 activation in the tumor microenvironment. + It promotes T cell and NK cell proliferation and recruits immune cells to infiltrate cold tumors by upregulating CXCL9 / CXCL10 chemokines. It has shown low systemic toxicity in a cynomolgus monkey model and has entered the preclinical translation stage. The PD-1 / IL-2α bispecific antibody IBI363 simultaneously blocks PD-1 signaling and activates IL-2α receptors, preferentially expanding effector T cells rather than Tregs, increasing the number of tumor-infiltrating lymphocytes and reducing TGF-β levels. Through synergistic dual-target remodeling of the cold tumor microenvironment, it achieved an objective response rate of 61.5% and a median duration of response of 19.3 months in a phase Ib clinical trial for melanoma, significantly superior to PD-1 monoclonal antibodies.

[0004] Interleukin-21 (IL-21) is a protein produced by the activation of CD4+. +IL-21, a pleiotropic cytokine secreted by T cells and NKT cells, regulates multiple aspects of the immune response through its receptor IL-21R and the JAK-STAT signaling pathway. IL-21 plays a central role in anti-tumor immunity: on the one hand, it directly activates CD8+ T cells, promoting effector functions (such as granzyme / perforin expression) through STAT3 signaling, while inhibiting the upregulation of exhaustion markers such as PD-1, thus prolonging the duration of the immune response; on the other hand, IL-21 significantly enhances the cytotoxicity and antibody-dependent killing function (ADCC) of natural killer (NK) cells, and synergistically enhances the survival and infiltration capacity of NK cells in the tumor microenvironment with IL-15. Furthermore, IL-21's ability to remodel the immune microenvironment is particularly crucial—by inhibiting the activity of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), antagonizing TGF-β-mediated immunosuppression, and simultaneously inducing the secretion of chemokines (CXCL9 / 10) to recruit effector immune cells, synergistically improving tumor angiogenesis abnormalities and stromal fibrosis, thereby breaking down the immune tolerance barrier. Currently, IL-21 has shown potential in clinical trials for melanoma, lymphoma, and other diseases.

[0005] CD40 ligand (CD40L) is a type II transmembrane protein member of the tumor necrosis factor (TNF) superfamily. It plays a crucial role in the activation of CD40 ligands. + CD40L is expressed on the surface of T cells, B cells, and certain cancer cells. By binding to the CD40 receptor on the surface of dendritic cells and other immune cells, CD40L activates downstream immune signaling pathways, thereby promoting antigen presentation and enhancing T cell function. After binding to CD40, CD40L triggers downstream signaling pathways such as NF-κB and MAPK, promoting dendritic cell maturation and antigen cross-presentation, and activating CD8+. + T-cell killing function and induction of tumor-associated macrophage (TAM) polarization from the pro-tumor M2 type to the anti-tumor M1 type, remodeling the immunosuppressive tumor microenvironment. Clinical studies have shown that CD40 agonists (such as Mitazalimab) combined with chemotherapy achieved an objective response rate (ORR) of 40.4% and a median overall survival (mOS) of 14.3 months in pancreatic cancer. The unique role of CD40L in cold tumor transformation and immune microenvironment reprogramming makes it an important area of ​​exploration for combination immunotherapy of tumors.

[0006] 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 protein expression efficiency is low and purification is difficult; linkers may introduce immunogenicity, and the difference in half-life between the two may affect pharmacokinetics and targeting; the risk of synergistic enhancement of immunity or induction of cytokine storm is difficult to predict. Summary of the Invention

[0007] 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 by fusing IL-21 with CD40L. This invention achieves functional synergy and complementarity between CD40L and IL-21 by constructing a CD40L-IL-21 fusion protein: CD40L activates APCs to initiate T cell immune responses, while IL-21 directly supports effector T cell expansion and memory maintenance, inhibits Treg function, and reverses T cell exhaustion, forming a "natural immune activation-adaptive immune maintenance" mechanism to synergistically break down the immunosuppressive barrier.

[0008] The present invention provides a fusion protein comprising a CD40L trimer or a variant thereof, and the cytokine IL-21 or a variant thereof; the CD40L trimer comprises three linked CD40L monomers.

[0009] In some embodiments, the three CD40L monomers in the CD40L trimer are linked by peptide linkers.

[0010] 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.

[0011] In some embodiments, the amino acid sequence of the peptide linker is shown in SEQ ID NO:5.

[0012] 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.

[0013] In some embodiments, the fusion protein further includes an Fc domain or a variant thereof; the Fc domain is, for example, IgG Fc.

[0014] 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).

[0015] In some embodiments, the fusion protein provided by the present invention includes at least (i) IL-21 or a variant thereof, (ii) an Fc domain or a variant thereof, and (iii) a CD40L trimer or a variant thereof.

[0016] 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-21 connected in sequence.

[0017] In some embodiments, the CD40L trimer is connected to the Fc domain via a first linker, and / or the Fc domain is connected to the cytokine IL-21 via a second linker.

[0018] In some embodiments, the amino acid sequence of the CD40L trimer is shown in SEQ ID NO:2.

[0019] In some implementations, the IgG Fc is derived from mice or humans, and / or the IgG Fc is IgG1 Fc.

[0020] In some embodiments, the IgG Fc is human IgG1 Fc; its amino acid sequence is preferably as shown in SEQ ID NO:9.

[0021] In some embodiments, the amino acid sequence of the cytokine IL-21 is shown in SEQ ID NO:8.

[0022] In some embodiments, the amino acid sequence of the first linker is shown in SEQ ID NO:6.

[0023] In some embodiments, the amino acid sequence of the second linker is shown in SEQ ID NO:7.

[0024] In some specific embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO:1.

[0025] In another aspect, the present invention provides an isolated nucleic acid that encodes the fusion protein provided by the present invention.

[0026] In some embodiments, the sequence of the isolated nucleic acid is shown in SEQ ID NO:11.

[0027] In another aspect, the present invention provides a recombinant vector comprising the isolated nucleic acid provided by the present invention.

[0028] In some implementations, the recombinant vector is a recombinant expression vector or a recombinant cloning vector.

[0029] In some implementations, the recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0030] 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.

[0031] In some embodiments, the mammalian expression vector is pcDNA3.4.

[0032] 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.

[0033] In some embodiments, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0034] In some embodiments, the eukaryotic cells are yeast cells or mammalian cells, such as CHO-S cells.

[0035] 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.

[0036] In some implementations, the fusion protein is purified from the culture product.

[0037] Another aspect of the present invention provides a pharmaceutical composition comprising the fusion protein provided by the present invention, and a pharmaceutically acceptable carrier.

[0038] 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.

[0039] 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.

[0040] 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:

[0041] 1) Activate the immune response in the tumor microenvironment;

[0042] 2) Activate the lymph node immune response;

[0043] 3) Promotes the maturation of human dendritic cells;

[0044] 4) Activator CD8 + T cells;

[0045] 5) Inhibits tumor cell growth; and,

[0046] 6) Enhance NK cell function.

[0047] In some implementation schemes, activating 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.

[0048] In some implementation schemes, activation of the tumor microenvironment immune response promotes CD8 + T cell infiltration and activation.

[0049] In some implementations, CD8 is promoted + T cell infiltration and activation upregulate CD8 in tumor tissue. + The proportion of T cells.

[0050] 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.

[0051] In some implementations, CD8 is promoted + T cell infiltration and activation enhance intratumoral CD8. + T cell effector functions, such as increasing IFN-γ secretion.

[0052] In some implementations, activating the lymph node immune response means enhancing the activation and function of immune cells in the inguinal lymph nodes.

[0053] 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.

[0054] 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 + Expression level of IFN-γ, an effector molecule of T cells

[0055] In some implementations, promoting human dendritic cell (DC) maturation is achieved by enhancing the activation and maturation of human DCs, for example by upregulating the expression levels of key activation / maturation markers CD80 and CD86 on the surface of human DCs.

[0056] In some implementation schemes, human CD8 is activated. + T cells promote activation and reverse depletion; for example, by increasing CD8. + The expression of CD69, a marker of T cell activation, and / or the expression of PD-1, a marker of T cell exhaustion, are reduced.

[0057] 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).

[0058] In some implementations, the tumor cells are solid tumors, such as melanoma cells.

[0059] 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.

[0060] 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.

[0061] Another aspect of the present invention provides a pillbox set, the pillbox set comprising pillbox A and pillbox B, wherein:

[0062] The medicine box A contains the fusion protein provided by the present invention or the pharmaceutical composition provided by the present invention.

[0063] 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.

[0064] In some implementations, the tumor is a solid tumor, such as melanoma.

[0065] 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.

[0066] In some implementations, the co-stimulation system further comprises an adjuvant.

[0067] 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.

[0068] 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.

[0069] In some implementations, the method is for non-diagnostic and non-therapeutic purposes.

[0070] 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.

[0071] In some implementations, the method is for non-diagnostic and non-therapeutic purposes.

[0072] 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.

[0073] In some implementations, the method is for non-diagnostic and non-therapeutic purposes.

[0074] 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.

[0075] In some implementations, the method is for diagnostic and therapeutic purposes.

[0076] In some implementations, the tumor cells are solid tumor cells, such as melanoma cells.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] In some implementations, the tumor is a solid tumor, such as melanoma.

[0082] 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.

[0083] In some implementations, the tumor is a solid tumor, such as melanoma.

[0084] 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.

[0085] 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.

[0086] The reagents and raw materials used in this invention are all commercially available.

[0087] The positive and progressive effects of this invention are as follows: The CD40L-IL-21 fusion protein constructed in this invention exhibits superior anti-tumor effects compared to the combined drug administration group (IL-21+CD40L group). It can activate the immune response in the tumor microenvironment, enhance tumor immune infiltration, and thus exert an anti-tumor effect; it can also increase the proportion of dendritic cells (DCs) in inguinal lymph nodes, and enhance their recruitment and activation of CD8+. + 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.

[0088] This invention constructs a CD40L-IL-21 fusion protein to achieve synergistic and complementary functions of CD40L and IL-21: CD40L activates APCs to initiate T cell immune responses, while IL-21 directly supports effector T cell expansion and memory maintenance, inhibits Treg function, and reverses T cell exhaustion, forming a "natural immune activation-adaptive immune maintenance" model to synergistically break down the immunosuppressive barrier. Attached Figure Description

[0089] Figure 1A This is a schematic diagram of the fusion protein structure.

[0090] Figure 1B This is an SDS-PAGE gel image of the fusion protein in both reduced and non-reduced states.

[0091] Figure 1C This is the HPLC analysis chromatogram of the fusion protein.

[0092] Figure 2 This demonstrates the in vivo antitumor effect of the fusion protein.

[0093] Figure 3A To enhance CD45 in mouse tumor-associated immune cells using fusion protein + Cell activation and function.

[0094] Figure 3B The fusion protein promotes CD8 in tumor tissue + Upregulation of the proportion of T cells.

[0095] Figure 3C The fusion protein promotes CD8 in tumor tissue + The expression level of CD69, a marker of T cell activation.

[0096] Figure 3D To enhance CD8 in tumor tissue with fusion protein + The amount of IFN-γ secreted, a marker of T cell killing function.

[0097] Figure 4 The fusion protein enhances the activation and function of dendritic cells in the mouse inguinal lymph nodes.

[0098] Figure 5A CD8 in inguinal lymph nodes after fusion protein treatment + The expression level of CD69, a marker of T cell activation.

[0099] 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.

[0100] Figure 6 The fusion protein significantly upregulated the expression levels of DC surface activation markers (CD80, CD86).

[0101] Figure 7 The fusion protein significantly increased CD8 + It reduced the expression of CD69, a marker of T cell activation, while simultaneously decreasing the expression of PD-1, a marker of T cell exhaustion.

[0102] Figure 8A CD8 was enhanced for the fusion protein + The level of granzyme B (GZMB), a functional effector molecule of T cells.

[0103] Figure 8B CD8 was enhanced for the fusion protein + The level of tumor necrosis factor-α (TNF-α), a functional effector molecule of T cells.

[0104] Figure 8C CD8 was enhanced for the fusion protein + The level of interferon-γ (IFN-γ), a functional effector molecule of T cells. Detailed Implementation

[0105] 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.

[0106] Example 1: Expression and purification of fusion protein

[0107] This embodiment provides a CD40L-IL-21 fusion protein (CD40L-Fc-IL-21), the structural diagram of which is shown below. Figure 1C 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-21. The CD40L trimer consists of three CD40L monomers covalently linked together by a peptide linker, and its amino acid sequence is as follows:

[0108] The amino acid sequence of the CD40L-IL-21 fusion protein (SEQ ID NO:1):

[0109] MGWSCIILFLVATATGVHSNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGGGGSGGGSQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGGGGSGGGSQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGGGGSGGGGSGGGGSASEPKSCDKTHTCPPCPAPELLGDDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGGSGGGGGSHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSCD Amino acid sequence of the 40L trimer (SEQ ID NO:2):

[0110] NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGGGGSGGGSQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGGGGSGGGSQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

[0111] Amino acid sequence of CD40L monomer (SEQ ID NO:3):

[0112] NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

[0113] Amino acid sequence of CD40L monomer (SEQ ID NO:4):

[0114] QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

[0115] Peptide linker: GGGGSGGGS (SEQ ID NO:5)

[0116] First linker: GGGGSGGGGSGGGGS (SEQ ID NO:6)

[0117] Second connector: GGGGSGGGGGSGGGGGS (SEQ ID NO:7)

[0118] The amino acid sequence of IL-21 (SEQ ID NO:8)

[0119] HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

[0120] The amino acid sequence of the Fc domain (SEQ ID NO:9)

[0121] ASEPKSCDKTHTCPPCPAPELLGDDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP RPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0122] This fusion protein was prepared through the following steps:

[0123] 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.

[0124] Target DNA sequence (SEQ ID NO:11):

[0125]

[0126] 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 ExpiCHO 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.

[0127] 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 the buffer into the final formulation buffer.

[0128] Figure 1A This is a schematic diagram of the fusion protein structure. The purified protein was analyzed using SDS-PAGE (…). Figure 1B ), HPLC Figure 1C The molecular weight and purity were determined by analysis.

[0129] Example 2: Fusion protein exhibits in vivo antitumor activity.

[0130] 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-21-FC group), a combination drug group (IL-21+CD40L group), and a fusion protein group (CD40L-IL-21 group). Seven days after subcutaneous tumor construction, the corresponding fusion 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.

[0131] Figure 2 The study revealed that the tumor volume in the fusion protein group (CD40L-IL-21 group) was significantly smaller than that in the single-drug control groups (CD40L-FC group and IL-21-FC group), indicating that the fusion protein has significant anti-tumor activity. Furthermore, the anti-tumor effect of the fusion protein group was slightly better than that of the combination therapy group (IL-21+CD40L group).

[0132] Example 3: Fusion protein enhances the activation and function of immune cells in mouse tumors.

[0133] 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.

[0134] Figures 3A-3D The results showed that after treatment with the CD40L-IL-21 fusion protein, CD45 in mouse melanoma tissues was reduced. + 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. Further analysis revealed CD8+ in the tumor tissue. + Upregulation of T cell ratio ( Figure 3B Furthermore, the expression level of its activation marker CD69 and the secretion of its cytotoxic function indicator IFN-γ were significantly enhanced. Figure 3C , Figure 3D This indicates that the CD40L-IL-21 fusion protein can enhance tumor immune infiltration, activate the immune response in the tumor microenvironment, and thus exert an anti-tumor effect.

[0135] Example 4: Fusion protein enhances the activation and function of immune cells in mouse inguinal lymph nodes

[0136] This study selected inguinal lymph nodes to elucidate the immunomodulatory mechanism induced by the fusion protein in mice, with particular focus on dendritic cells (cDCs) and CD8+. + T cell interactions, and the activation of CD8 by dendritic cells through antigen presentation. + The role of T cells. In this experimental example, inguinal lymph nodes from tumor-bearing mice in Experiment 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 (BDPharm Lyse) 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, 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 finally resuspended in FACS buffer and filtered. ≥10,000 CD45 cells were collected by flow cytometry. + Cells, and then analyzed DCs and CD8 + The state of T-cell immune activation.

[0137] Figure 4 The results showed that treatment with the CD40L-IL-21 fusion protein significantly increased the proportion of dendritic cells (DCs) in the inguinal lymph nodes of mice, accompanied by upregulation of activation markers. Figure 4 ). Figures 5A-5B The results showed that CD8+ in inguinal lymph nodes were reduced after treatment with the CD40L-IL-21 fusion protein. + The expression levels of CD69, a marker of T cell activation, and the expression of the effector molecule interferon-γ (IFN-γ) were upregulated. Figure 5A , Figure 5BThe CD40L-IL-21 fusion protein can increase the proportion of dendritic cells (DCs) in inguinal lymph nodes and enhance their recruitment and activation of CD8+. + The ability of T cells is enhanced, thereby exhibiting the dual activation effect of the fusion protein and further exerting its anti-tumor effect.

[0138] Example 5: Fusion protein enhances the activation and function of human immune cells

[0139] In this study, peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy volunteers using Ficoll density gradient centrifugation, and two 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. 5 Cells were added at densities of 500 ng / mL CD40L-IL-2 fusion protein or phosphate-buffered saline (PBS) to 24-well plates (24-well plates, 500 μL) for 24 hours. After treatment, pretreatment with FcX inhibitor (BioLegend) was performed, followed by detection of the mean fluorescence intensity (MFI) of CD86 and CD80 surface markers. The experiment was repeated three times, with each experiment performed in three independent technical replicates. Next, CD8... + T cell functional response assay: PBMCs were administered at a concentration of 3 × 10⁻⁶ 5 Cells were seeded at a density in 96-well plates and treated with the same dosage for 24 hours. Following treatment, surface staining (CD45 / CD3 / CD8 / CD69) and intracellular staining (GZMB, TNF-α, and IFN-γ) were performed. Flow cytometry was used to analyze CD3+. + CD8 + The positivity rates of various indicators in the T cell population were measured, and the antibodies used were all purchased from BioLegend.

[0140] As in this embodiment Figures 6-8C As shown, after treatment with the CD40L-IL-21 fusion protein, DCs and CD8... + The expression levels of T cell activation and function-related markers were significantly increased. Figure 6 As shown, the CD40L-IL-21 fusion protein significantly upregulated the expression levels of DC surface activation markers (CD80, CD86), indicating that the fusion protein can effectively promote the in vitro activation of DCs. Figure 7 As shown, the CD40L-IL-21 fusion protein significantly enhanced CD8 +The expression of CD69, a marker of T cell activation, was reduced, while CD8 expression was decreased. + Expression of PD-1, a marker of T cell exhaustion. Figures 8A-8C The results showed that the CD40L-IL-21 fusion protein enhanced CD8. + T cell functional effector molecules (including granzyme B (GZMB), Figure 8A ), tumor necrosis factor-α (TNF-α, Figure 8B ) and interferon-γ (IFN-γ, Figure 8C The level of CD8 demonstrates that the fusion protein can effectively enhance CD8 levels. + The CD40L-IL-21 fusion protein effectively activates these cells, exhibiting superior performance compared to single-drug regimens.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises a CD40L trimer or a variant thereof, and the cytokine IL-21 or a variant thereof; the CD40L trimer comprises three linked CD40L monomers; The amino acid sequence of the fusion protein is shown in SEQ ID NO:

1.

2. An isolated nucleic acid, characterized in that, The isolated nucleic acid encodes the fusion protein as described in claim 1.

3. The isolated nucleic acid as described in claim 2, characterized in that, The sequence of the isolated nucleic acid is shown in SEQ ID NO:

11.

4. A recombinant vector, characterized in that, The recombinant vector contains the isolated nucleic acid as described in claim 2 or 3.

5. The recombinant vector as described in claim 4, characterized in that, The recombinant vector is a recombinant expression vector or a recombinant cloning vector.

6. The recombinant vector as described in claim 5, characterized in that, The recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

7. The recombinant vector as described in claim 5, characterized in that, The prokaryotic expression vectors were selected from Escherichia coli expression vectors, Bacillus subtilis expression vectors, and Streptomyces expression vectors; the eukaryotic expression vectors were selected from yeast expression vectors, insect expression vectors, and mammalian expression vectors.

8. The recombinant vector as described in claim 7, characterized in that, The mammalian expression vector is pcDNA3.

4.

9. A transformant, characterized in that, The transformant comprises the isolated nucleic acid as described in claim 2 or 3 or the recombinant vector as described in any one of claims 4-8, wherein the transformant is a non-animal variety and a non-plant variety.

10. The transformant as described in claim 9, characterized in that, The host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

11. The transformant as described in claim 10, characterized in that, The eukaryotic cells are yeast cells or mammalian cells.

12. The transformant as described in claim 11, characterized in that, The eukaryotic cells are CHO-S cells.

13. 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 9-12, and obtaining the fusion protein from the culture.

14. The method as described in claim 13, characterized in that, The fusion protein was purified from the culture product.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the fusion protein as described in claim 1, and a pharmaceutically acceptable carrier.

16. The use of the fusion protein of claim 1, the isolated nucleic acid of claim 2 or 3, the recombinant vector of any one of claims 4-8, the transformant of any one of claims 9-12, or the pharmaceutical composition of claim 15 in the preparation of a product that inhibits the growth of melanoma cells.

17. A reagent kit, characterized in that, The kit contains the fusion protein as described in claim 1 or the pharmaceutical composition as described in claim 15.

18. The kit according to claim 17, 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.

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