Dimer compound of novel protein complex, preparation method therefor, and pharmaceutical use thereof

Abstract

The present invention relates to the technical field of biology, and disclosed are a dimer compound of a novel protein complex, a preparation method therefor, and a pharmaceutical use thereof. A dimeric fusion protein provided by the present invention comprises an IL-2 / IL-15 protein chimera and an IL-15Rα Fc fusion protein. The IL-2 / IL-15 protein chimera comprises a polypeptide from the N-terminus of IL-2 and a full-length peptide chain of IL-15 or a variant thereof. The IL-15Rα Fc fusion protein comprises an IL-15Rα Sushi domain and an Fc CH2-CH3 domain. The dimeric fusion protein of the present invention can mediate activation and proliferation of immune cells and can be used for treating tumor diseases.

Description

A novel protein complex dimer compound, its preparation method and pharmaceutical applications

[0001] This invention claims priority to Chinese Patent Application No. 2024115350779, filed on October 30, 2024, entitled "Novel IL15 Agonist and its Preparation Method and Pharmaceutical Use", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biotechnology, specifically to a dimer compound comprising an IL-2 / IL-15 protein chimera and an IL-15Ra Fc fusion protein complex, its preparation method, and its pharmaceutical use. Background Technology

[0003] Interleukins (ILs) are a family of cytokines primarily expressed and secreted by leukocytes, playing a crucial role in promoting the proliferation, differentiation, and immune regulation of immune cells such as T cells and NK cells. Most interleukin cytokines exert multiple roles in anti-tumor activity. Within this cytokine family, interleukin-15 (IL-15) shares a common gamma (γc) chain receptor with other members of the IL-2 protein family (such as IL-2, IL-4, IL-7, IL-9, and IL-21). It can promote innate and adaptive immune responses by stimulating CD8+ / CD4+ T cells and natural killer (NK) cells without activating regulatory T (Treg) cells or causing the death of effector T cells and NK cells.

[0004] Numerous studies have explored using gene editing technology to arm immune cells with the co-expression of one or more interleukins (such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-23) or in combination with their receptors for anti-cancer purposes. Among these, interleukin-2 (IL-2) is the most thoroughly investigated drug and has been approved for cancer treatment. Patients with metastatic melanoma and renal cell carcinoma have experienced regression and durable remission of metastatic tumors after using IL-2. In vitro expansion of IL-2 can promote the activation and proliferation of immune cells, including tumor-infiltrating T cells, CAR-T cells, and NK cells. However, the higher degree of differentiation of immune cells resulting from IL-2 expansion may affect their long-term persistence and survival.

[0005] Because IL-15 is functionally similar to IL-2 and possesses several significant advantages, including lack of stimulation of regulatory T cell populations, low activation-induced cell death (AICD), and lower toxicity, these properties have led to IL-15 being listed as one of the most promising cytokines for cancer immunotherapy. In addition to its use alone in cancer immunotherapy, IL-15 has been incorporated into many adoptive cell therapies for cancer treatment, particularly in combination with chimeric antigen receptor (CAR) engineering. For example, IL-15-armed CAR T cells, NK cells, and unconventional T cells can proliferate and activate NK cells, CD8+ cytotoxic T cells, and CD4+ helper T cells. The proliferation of these key cancer-killing immune cells, coupled with the activation of memory-enabled T cells, generates a durable response of the human immune system to IL-15 drugs. This durable response, through long-term memory, kills tumor cells, forming the basis for the development of cancer vaccines.

[0006] However, natural IL-15 suffers from significant drug-likeness issues, namely low expression levels, short half-life, and poor marketability. Therefore, to develop IL-15-based drugs, it is essential to enhance its expression levels, prolong its half-life, and improve its potency. Numerous studies have shown that complexes formed from the Sushi domain of IL-15 and soluble IL-15Ra can increase IL-15 expression and are significantly more potent than IL-15 in stimulating the proliferation of memory CD8+ T lymphocytes and NK cells, as well as maintaining the viability of memory CD8+ T cells through transpresentation mechanisms. This indicates that creating IL-15 / Sushi domain fusion proteins can significantly improve efficacy.

[0007] In terms of prolonging half-life, the most common methods are increasing the molecular weight to reduce renal elimination or by targeting nascent Fc receptors. Molecules that have been tested include IL-15Ra-linker-IL-15 (RLI), N-803 (ALT-803), and NKTR255. N-803 (trade name: Anktiva) has recently been approved for the treatment of non-muscle-invasive bladder cancer (NMIBC) carcinoma in situ (CIS) that is unresponsive to BCG.

[0008] Another way to extend the half-life is to increase the glycosylation of the protein. For example, the long-acting erythropoietin drug Darbepoetin a (Aranesp) adds two glycosylation sites to the erythropoietin (EPO) molecule, making its half-life in the body longer than that of EPO and requiring a smaller dosage.

[0009] Although IL-15 and IL-2 are extremely similar in function and structure, IL-2 is expressed at high levels while IL-15 is expressed at low levels. IL-15 expression is controlled at the transcriptional, translational, and intracellular transport levels, and structural differences between IL-15 and IL-2 also affect their expression levels. Compared to IL-2, IL-15 lacks a polypeptide containing multiple amino acid residues at its N-terminus, and this polypeptide also has an O-glycosylation site. Therefore, this invention forms a chimeric complex of IL-2 and IL-15, which is then combined with an IL-15Rα-Fc CH2-CH3 fusion protein to form a dimer fusion protein. Compared to the N-803 dimer fusion protein, the dimer fusion protein of this invention has higher expression levels, more glycosylation, resulting in a longer half-life, better in vivo biological activity, and druggability. Summary of the Invention

[0010] The purpose of this invention is to provide a novel dimeric fusion protein comprising an IL-2 / IL-15 protein chimera and an IL-15Ra Fc fusion protein, its preparation method, and its pharmaceutical applications.

[0011] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0012] On the one hand, the present invention provides a novel dimer fusion protein, wherein the dimer fusion protein comprises an IL-2 / IL-15 protein chimera and an IL-15RαFc fusion protein, wherein the IL-2 / IL-15 protein chimera comprises a polypeptide segment at the N-terminus of IL-2 and a full-length peptide chain of IL-15 or a variant thereof, and the IL-15RαFc fusion protein comprises an IL-15RαSushi domain and an Fc CH2-CH3 domain;

[0013] The IL-2 / IL-15 protein chimera and IL-15RαFc fusion protein are QHJ-02V03, QHJ-02V05, QHJ-02V13, QHJ-02V15, and QHJ-02M02.

[0014] Specifically:

[0015] (1) QHJ-02V03: The IL-2 / IL-15 protein chimera has the sequence shown in SEQ ID NO:7, and the IL-15RαFc fusion protein has the sequence shown in SEQ ID NO:6; or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7 or 6; or;

[0016] (2) QHJ-02V05: The IL-2 / IL-15 protein chimera has the sequence shown in SEQ ID NO:8, and the IL-15RαFc fusion protein has the sequence shown in SEQ ID NO:6; or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:8 or 6;

[0017] (3) QHJ-02V13: The IL-2 / IL-15 protein chimera has the sequence shown in SEQ ID NO:9, and the IL-15RαFc fusion protein has the sequence shown in SEQ ID NO:6; or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:9 or 6; or;

[0018] (4) QHJ-02V15: The IL-2 / IL-15 protein chimera has the sequence shown in SEQ ID NO:9, and the IL-15RαFc fusion protein has the sequence shown in SEQ ID NO:10; or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:9 or 10;

[0019] (5)QHJ-02M02: The IL-2 / IL-15 protein chimera has the sequence shown in SEQ ID NO:9, and the IL-15RαFc fusion protein has the sequence shown in SEQ ID NO:11; or an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:9 or 11.

[0020] The IL-2 / IL-15 chimeric sequence of QHJ-02V13 is completely identical to the IL-2 / IL-15 chimeric sequence of QHJ-02M02, and the IL-15RαFc fusion protein sequence of QHJ-02V13 has 97.4% identity with the IL-15RαFc fusion protein sequence of QHJ-02M02.

[0021] The IL-2 / IL-15 chimeric sequence of QHJ-02V15 is completely identical to the IL-2 / IL-15 chimeric sequence of QHJ-02M02, and the IL-15RαFc fusion protein sequence of QHJ-02V15 has 95.7% identity with the IL-15RαFc fusion protein sequence of QHJ-02M02.

[0022] Specifically, the IL-2 / IL-15 protein chimera has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO:7.

[0023] Specifically, the IL-15RαFc fusion protein has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO:6.

[0024] Specifically, the IL-2 / IL-15 protein chimera has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO:8.

[0025] Specifically, the IL-2 / IL-15 protein chimera has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO:9.

[0026] Specifically, the IL-15RαFc fusion protein has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO:10.

[0027] Specifically, the IL-15RαFc fusion protein has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO:11.

[0028] Specifically, the IL-2 / IL-15 protein chimera is a fusion of a polypeptide with glycosylation sites of IL-2 and recombinant human IL-15 (rhIL-15). It has a larger molecular weight than IL15 protein and more glycosylations on the protein. The cellular expression level of the IL-2 / IL-15 protein chimera is higher than that of IL15 protein.

[0029] Specifically, the IL-2 polypeptide consists of 5 to 30 amino acids.

[0030] More preferably, the polypeptide segment consists of 6 to 15 amino acids.

[0031] More preferably, the polypeptide segment contains one or more glycosylation sites.

[0032] More preferably, the polypeptide segment has at least 60% sequence identity with APTSSSTKKTQL.

[0033] Preferably, the polypeptide segment is APTSSSTKKTQL, which consists of 12 amino acids at the N-terminus of natural IL-2.

[0034] SEQ ID NO:7:

[0035] SEQ ID NO:6:

[0036] SEQ ID NO:8:

[0037] SEQ ID NO:9:

[0038] SEQ ID NO:10:

[0039] SEQ ID NO:11:

[0040] In another aspect, the present invention provides a method for preparing a novel dimer fusion protein, comprising the following steps:

[0041] (1) Construct plasmids expressing IL-2 / IL-15 chimera and IL-15RαFc;

[0042] (2) Culture the cells, add the IL-2 / IL-15 chimeric plasmid DNA and IL-15Ra Fc plasmid DNA solution to the culture medium, and add the transfection reagent for transfection;

[0043] (3) Add transfection enhancer, culture, centrifuge, and collect protein supernatant.

[0044] Specifically, the cell culture conditions in step (2) are 37°C, 80% relative humidity, and 8% CO2.

[0045] Specifically, the culture medium for cell culture contains penicillin / streptomycin.

[0046] Specifically, the cells are selected from any one of Expi293F cells, CHO cells, and HEK293 cells.

[0047] Furthermore, the cells mentioned are Expi293F cells.

[0048] Specifically, the culture medium used for cell culture is selected from any one of BalanceCD 293 medium, MRS medium, M17 medium, PDA medium, LB medium, TB medium, SOB medium, MEM medium, DMEM medium, and RPMI 1640 medium.

[0049] Furthermore, the culture medium used for the cell culture is BalanceCD 293 medium.

[0050] Specifically, in step (2), the ratio of IL-2 / IL-15 chimeric plasmid to IL-15RαFc plasmid is (1-5):1;

[0051] Furthermore, in step (2), the ratio of IL-2 / IL-15 chimeric plasmid to IL-15RαFc plasmid is 1:1 or 5:1;

[0052] Furthermore, in step (2), the ratio of the IL-2 / IL-15 chimeric plasmid to the IL-15RαFc plasmid is 1:1.

[0053] Specifically, in step (2), the total amount of plasmid is 100 μg / 100 mL of cells.

[0054] Specifically, the transfection time in step (2) is 16-20 hours; further, the transfection time is 20 hours.

[0055] Specifically, the transfection enhancer mentioned in step (3) includes VGlucose and VVPA; further, the ratio of VGlucose to VVPA is (5-8):1; and even further, the ratio of VGlucose to VVPA is 7:1.

[0056] Specifically, the concentration of VGlucose is 400 g / L, and the concentration of VVPA is 100 g / 1.5 L.

[0057] Specifically, the amount of transfection enhancer added is 20 ml / L of cells.

[0058] Specifically, the centrifugation conditions described in step (3) are 4°C, 2000-8000 rpm for 5-10 min.

[0059] In another aspect, the present invention provides a nucleic acid molecule that encodes the aforementioned novel dimer fusion protein.

[0060] Specifically, when the dimer fusion protein is QHJ-02V03, the ORF sequence encoding the IL-2 / IL-15 chimera is shown in SEQ ID NO:12; the ORF sequence encoding IL-15Ra Fc is shown in SEQ ID NO:13; or a sequence having at least 70% sequence identity with the sequences shown in SEQ ID NO:12-13;

[0061] Specifically, when the dimer fusion protein is QHJ-02V05, the ORF sequence encoding the IL-2 / IL-15 chimera is shown in SEQ ID NO:14; the ORF sequence encoding IL-15Ra Fc is shown in SEQ ID NO:13; or a sequence having at least 70% sequence identity with the sequences shown in SEQ ID NO:13-14.

[0062] Specifically, when the dimer fusion protein is QHJ-02V13, the ORF sequence encoding the IL-2 / IL-15 chimera is shown in SEQ ID NO:15; the ORF sequence encoding IL-15Ra Fc is shown in SEQ ID NO:13; or a sequence having at least 70% sequence identity with the sequences shown in SEQ ID NO:13 and SEQ ID NO:15.

[0063] Specifically, when the dimer fusion protein is QHJ-02V15, the ORF sequence encoding the IL-2 / IL-15 chimera is shown in SEQ ID NO:15; the ORF sequence encoding IL-15Ra Fc is shown in SEQ ID NO:16; or a sequence having at least 70% sequence identity with the sequences shown in SEQ ID NO:15-16.

[0064] Specifically, when the dimer fusion protein is QHJ-02M02, the ORF sequence encoding the IL-2 / IL-15 chimera is shown in SEQ ID NO:15; the ORF sequence encoding IL-15Ra Fc is shown in SEQ ID NO:17; or a sequence having at least 70% sequence identity with the sequences shown in SEQ ID NO:15 and SEQ ID NO:17.

[0065] SEQ ID NO:12

[0066] SEQ ID NO:13

[0067] SEQ ID NO:14:

[0068] SEQ ID NO:15:

[0069] SEQ ID NO:16:

[0070] SEQ ID NO:17:

[0071] In another aspect, the present invention provides an expression vector carrying the aforementioned nucleic acid molecules.

[0072] Specifically, the expression vector is a eukaryotic expression vector.

[0073] Furthermore, the expression vector is a plasmid expression vector.

[0074] In another aspect, the present invention provides a host cell comprising: the nucleic acid molecule described above; or: the expression vector described above.

[0075] Specifically, the host cells include eukaryotic cells.

[0076] Furthermore, the host cells are derived from mammalian cells, insect cells, and yeast cells.

[0077] Furthermore, the mammalian cells mentioned include CHO cells and HEK293 cells.

[0078] In another aspect, the present invention provides the use of the above-mentioned novel dimer fusion protein, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, or the above-mentioned host cell in the preparation of a drug for treating cancer.

[0079] Furthermore, the cancers mentioned include melanoma, lung cancer, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, T-cell lymphoma, B-cell lymphoma, liver cancer, kidney cancer, small cell lung cancer, non-small cell lung cancer, nasopharyngeal carcinoma, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, acute myeloid leukemia, cervical cancer, thyroid cancer, lymphoma, neuroblastoma, brain tumor, myeloma, and glioma.

[0080] In another aspect, the present invention provides the use of the above-mentioned novel dimer fusion protein, or the above-mentioned nucleic acid molecule, or the above-mentioned expression vector, or the above-mentioned host cell in the preparation of a drug for inhibiting lung metastasis of melanoma.

[0081] In another aspect, the present invention provides a pharmaceutical composition comprising the novel dimer fusion protein described above, the nucleic acid molecule described above, the expression vector described above, or the host cell described above.

[0082] Specifically, the pharmaceutical composition is used to treat cancer.

[0083] Furthermore, the cancers mentioned include melanoma, lung cancer, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, T-cell lymphoma, B-cell lymphoma, liver cancer, kidney cancer, small cell lung cancer, non-small cell lung cancer, nasopharyngeal carcinoma, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, acute myeloid leukemia, cervical cancer, thyroid cancer, lymphoma, neuroblastoma, brain tumor, myeloma, and glioma.

[0084] Specifically, the pharmaceutical composition may also include a pharmaceutically acceptable carrier.

[0085] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of the following: excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, and flavoring agents.

[0086] Specifically, the dosage form of the drug, depending on the route of administration, includes, but is not limited to, gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0087] Furthermore, the gastrointestinal dosage forms include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0088] Furthermore, the non-gastrointestinal dosage forms include, but are not limited to: injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0089] Furthermore, the injectable dosage forms include, but are not limited to, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections.

[0090] Furthermore, the respiratory drug delivery formulations include, but are not limited to, sprays, aerosols, and powder inhalers.

[0091] Furthermore, the skin delivery dosage forms include, but are not limited to, lotions, ointments, topical solutions, styrosine, pastes, and patches.

[0092] Furthermore, the mucosal drug delivery dosage forms include, but are not limited to, eye drops, nasal drops, ophthalmic ointments, sublingual tablets, and patches.

[0093] Furthermore, the cavity drug delivery dosage forms include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, and pellets.

[0094] Preferably, the dosage form of the drug is an injectable dosage form.

[0095] More preferably, the injectable dosage form is a subcutaneous injection.

[0096] Specifically, the pharmaceutical composition can be used in combination with anticancer drugs for treatment.

[0097] Furthermore, the composition includes, but is not limited to, BCG, vemurafenib tablets, dacarbazine, temozolomide capsules, paclitaxel, cisplatin, carboplatin, and formustin.

[0098] The beneficial effects of this invention are as follows:

[0099] (1) The novel dimer fusion protein described in this invention has a higher protein expression level.

[0100] (2) The novel dimer fusion protein of the present invention has more glycosyl groups.

[0101] (3) The present invention provides a novel dimer fusion protein that can significantly reduce the tumor volume and tumor area in melanoma mice, and has a good therapeutic effect on melanoma. Furthermore, the dimer fusion protein of the present invention can effectively inhibit melanoma lung metastasis.

[0102] Terminology Explanation

[0103] Unless otherwise stated, the terminology used in this invention has the meanings commonly understood by one of ordinary skill in the art.

[0104] In this invention, the term "IL-2" or "IL2" is also known as T cell growth factor, a pleiotropic cytokine with the following functions: (1) activating T cells and promoting cytokine production; (2) stimulating NK cell proliferation, enhancing NK cell killing activity and producing cytokines, and inducing LAK cell production; (3) promoting B cell proliferation and antibody secretion; and (4) activating macrophages. The "IL-2" described in this invention can be any IL-2 or its mutants, such as human IL-2, non-human mammalian IL-2, or non-mammal IL-2. Exemplary non-human mammals include pigs, rabbits, monkeys, chimpanzees, and mice, while non-mammals include chickens.

[0105] In this invention, the term "IL-15" or "IL15" refers to a pleiotropic cytokine that activates T cells, B cells, and NK cells, and mediates their proliferation and survival. Furthermore, IL-15 can activate, maintain, and expand CD8+ memory T cells. The "IL-15" described in this invention can be any IL-15 or its mutants, such as human IL-15, non-human mammalian IL-15, or non-mammal IL-15. Exemplary non-human mammals include pigs, rabbits, monkeys, chimpanzees, and mice; non-mammals include chickens.

[0106] In this invention, the term "IL-15Ra" can refer to IL-15Ra or its functional fragments from any species, such as human IL-15Ra, non-human mammalian IL-15Ra, or non-mammal IL-15Ra. Exemplary non-human mammals include pigs, rabbits, monkeys, orangutans, and rats, while non-mammals include chickens.

[0107] In this invention, the term "IL-15Ra-like protein" refers to a functional mutant of IL-15Ra formed by the deletion, insertion, or mutation of one or more amino acids, possessing the ability to bind to its ligand molecule such as IL15. Preferably, it is a human IL-15Ra molecule, more preferably an extended form of the IL-15Ra extracellular domain segment, i.e., a molecule with human IL-15Ra activity obtained by inserting one or more amino acid mutations starting from the C-terminus of the extracellular domain segment. IL-15Ra Fc refers to an IL-15Ra Fc fusion protein, including the IL-15RαSushi domain and the Fc CH2-CH3 domain.

[0108] In this invention, the term "Fc region" refers to the constant region of an immunoglobulin chain, particularly the carboxyl terminus or a portion thereof of the constant region of the immunoglobulin heavy chain. It lacks antigen-binding activity and is the site where antibody molecules interact with effector molecules and cells. The "immunoglobulin Fc region" described in this invention can be any Fc or its variants, derived from humans or non-human mammals. For example, the immunoglobulin Fc region may include a combination of two or more domains of the heavy chain CH1, CH2, CH3, and CH4 with the immunoglobulin hinge region. Fc can originate from different species, preferably human immunoglobulins. Based on the amino acid sequence of the heavy chain constant region, immunoglobulins can be classified into different types, mainly five classes: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, IgG-4; IgA-1 and IgA-2. The "Fc region" preferably includes at least one immunoglobulin hinge region, as well as the CH2 and CH3 domains of IgG. More preferably, it includes a CH2 domain, a CH3 domain, and an immunoglobulin hinge region of IgG1, wherein the starting amino acid position of the hinge region can be varied.

[0109] In this invention, the term "sequence identity" refers to the percentage of amino acid (or nucleotide) residues in a candidate sequence that are identical to those in a reference sequence after sequence alignment and the introduction of gaps (if necessary) to achieve maximum percentage sequence identity. For the purpose of determining percentage sequence identity, alignment can be performed in a variety of ways well known to those skilled in the art, such as using publicly available computer software like BLAST or ALIGN.

[0110] In this invention, the term "vector" includes nucleic acid vectors, such as DNA vectors (e.g., plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). The expression vectors of this invention contain polynucleotide sequences and additional sequence elements for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells.

[0111] In this invention, the term "treatment" refers to surgical or pharmaceutical procedures aimed at preventing, slowing (reducing) undesirable physiological changes or lesions in the treated subject, such as the progression of cancer (including melanoma). Beneficial clinical effects include, but are not limited to, symptom relief, disease severity reduction, disease stabilization, delay or slowing of disease progression, improvement or mitigation of the disease state, and remission.

[0112] In this invention, the term "pharmaceutical composition" refers to a composition containing one or more of the agonists or pharmaceutically acceptable carriers described in this invention. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and enabling it to exert its biological activity. Attached Figure Description

[0113] Figure 1 is a schematic diagram illustrating the general formula for constructing the novel dimer fusion protein of the present invention. The dimer fusion protein comprises an IL-2 / IL-15 chimeric protein and an IL-15Ra Fc fusion protein. The IL-2 / IL-15 chimeric protein consists of a peptide chain from the N-terminus of IL-2, followed by the full-length peptide chain of IL-15 or a variant thereof. The IL-15Ra Fc fusion protein includes an IL-15RaSushi domain and an Fc CH2-CH3 domain. The IL-15Ra Sushi domain and the CH2 domain are linked by a hinge containing three cysteine ​​residues. The IL-2 / IL-15 chimeric protein and the IL-15Ra Fc fusion protein form a complex, and the two complexes mainly form a homodimeric fusion protein through interactions between the CH3 domains and disulfide bonds in the hinge region.

[0114] Figure 2 shows the plasmids expressing IL15 and IL-15Ra Fc corresponding to the QHJ-02V02 (N-803 reference) polymer.

[0115] Figure 3 shows the plasmids corresponding to the QHJ-02V03 polymer that express IL-2 / IL-15 chimera and IL-15Ra Fc.

[0116] Figure 4 shows the plasmids corresponding to the QHJ-02V05 polymer that express IL-2 / IL-15 chimera and IL-15Ra Fc.

[0117] Figure 5 shows the plasmids corresponding to the QHJ-02V13 polymer that express IL-2 / IL-15 chimera and IL-15Ra Fc.

[0118] Figure 6 shows the plasmids corresponding to the QHJ-02V15 polymer that express IL-2 / IL-15 chimera and IL-15Ra Fc.

[0119] Figure 7 shows the plasmids corresponding to the QHJ-02M02 polymer that express IL-2 / IL-15 chimera and IL-15Ra Fc.

[0120] Figure 8 shows the gel electrophoresis diagram of QHJ-02V02 protein after purification by column A; in the figure, M: Marker; S: Supernatant of medium after expression; FT: Flow through after pulldown; E0.1M Glycine: Elute fraction with 0.1M Glycine.

[0121] Figure 9 shows the SDS-PAGE gel electrophoresis image of the QHJ-02V02 protein obtained after treatment with PNGase F and N-glycosidase F.

[0122] Figure 10 shows the gel electrophoresis diagram of the QHJ-02V03 protein after purification by column A.

[0123] Figure 11 shows the SDS-PAGE gel electrophoresis image of the QHJ-02V03 protein obtained after treatment with PNGase F and N-glycosidase F.

[0124] Figure 12 shows the gel electrophoresis diagram of the QHJ-02V05 protein after purification by column A.

[0125] Figure 13 shows the purification results of QHJ-02V02 via a QHP (5ml) column.

[0126] Figure 14 shows the SDS-PAGE detection results of QHJ-02V02 after purification by QHP column.

[0127] Figure 15 shows the purification results of QHJ-02V02#1 through an SEC column (Superdex 200 10 / 300).

[0128] Figure 16 shows the SDS-PAGE detection results of QHJ-02V02#1 after passing through the SEC column.

[0129] Figure 17 shows the purification results of QHJ-02V02#2 through an SEC column (Superdex 200 10 / 300).

[0130] Figure 18 shows the SDS-PAGE detection results of QHJ-02V02#2 after passing through the SEC column.

[0131] Figure 19 shows the SDS-PAGE purity analysis results of QHJ-02V02.

[0132] Figure 20 shows the SEC analysis column detection results of peak QHJ-02V02#1.

[0133] Figure 21 shows the SEC analysis column detection results of peak QHJ-02V02#2.

[0134] Figure 22 shows the complete protein spectrum analysis results of peak QHJ-02V02#1.

[0135] Figure 23 shows the complete protein spectrum analysis results of peak QHJ-02V02#2.

[0136] Figure 24 shows the purification results of QHJ-02V03 via a QHP (5ml) column.

[0137] Figure 25 shows the SDS-PAGE detection results of QHJ-02V03.

[0138] Figure 26 shows the purification results of QHJ-02V03#1 through an SEC column (Superdex 200 10 / 300).

[0139] Figure 27 shows the SDS-PAGE test results of QHJ-02V03#1 after passing through the SEC column.

[0140] Figure 28 shows the purification results of QHJ-02V03#2 through an SEC column (Superdex 200 10 / 300).

[0141] Figure 29 shows the SDS-PAGE test results of QHJ-02V03#2 after passing through the SEC column.

[0142] Figure 30 shows the SDS-PAGE purity analysis results of QHJ-02V03.

[0143] Figure 31 shows the SEC analysis column detection results of peak QHJ-02V03#1.

[0144] Figure 32 shows the SEC analysis column detection results of peak QHJ-02V03#2.

[0145] Figure 33 shows the complete protein spectrum analysis results of peak QHJ-02V03#1.

[0146] Figure 34 shows the complete protein spectrum analysis results of peak QHJ-02V03#2.

[0147] Figure 35 shows the results of IL-15 (Acro, #IL5-H5215) cell experiments.

[0148] Figure 36 shows the experimental results of QHJ02-V02 (#1 peak) cells.

[0149] Figure 37 shows the experimental results of QHJ02-V02 (#2 peak) cells.

[0150] Figure 38 shows the experimental results of QHJ02-V03 (#1 peak) cells.

[0151] Figure 39 shows the experimental results of QHJ02-V03 (#2 peak) cells.

[0152] Figure 40 shows the changes in tumor volume in mice during drug administration in each group.

[0153] Figure 41 shows the statistical graph of tumor volume in each group of mice.

[0154] Figure 42 shows the changes in average body weight of the animals in each group.

[0155] Figure 43 shows the average body temperature changes of the animals in each group.

[0156] Figure 44 shows the average number of lung metastases in each group of animals; the tumor weight of each treatment group (G2-G6) was statistically analyzed with that of the control group (G1), *P<0.05, **P≤0.01, ***P≤0.001.

[0157] Figure 45 shows lung metastasis lesions in group G1 animals (n=7). The small black dots in the lungs are lung metastasis lesions of B16F10 cells. Both sides of the lungs of each animal were photographed.

[0158] Figure 46 shows lung metastasis lesions in group G2 animals (n=7). The small black dots in the lungs are lung metastasis lesions of B16F10 cells. The lungs of each animal were photographed from both the front and back.

[0159] Figure 47 shows lung metastasis lesions in group G3 animals (n=7). The small black dots in the lungs are lung metastasis lesions of B16F10 cells. The lungs of each animal were photographed from both the front and back.

[0160] Figure 48 shows lung metastatic lesions in group G4 animals (n=7). The small black dots in the lungs are lung metastatic lesions of B16F10 cells. Both sides of the lungs of each animal were photographed.

[0161] Figure 49 shows lung metastasis lesions in group G5 animals (n=7). The small black dots in the lungs are lung metastasis lesions of B16F10 cells. The lungs of each animal were photographed from both the front and back.

[0162] Figure 50 shows lung metastatic lesions in group G6 animals (n=7). The small black dots in the lungs are lung metastatic lesions of B16F10 cells. Both sides of the lungs of each animal were photographed.

[0163] Figure 51 shows the gel electrophoresis diagram of the QHJ-02V13 protein after purification by column A.

[0164] Figure 52 shows the purification results of QHJ-02V13 through an SEC column (Superdex 200 10 / 300).

[0165] Figure 53 shows the gel electrophoresis diagram of the QHJ-02V15 protein after purification by column A.

[0166] Figure 54 shows the purification results of QHJ-02V15 through an SEC column (Superdex 200 10 / 300).

[0167] Figure 55 shows the gel electrophoresis diagram of the QHJ-02M02 protein after purification by column A.

[0168] Figure 56 shows the purification results of QHJ-02M02 through an SEC column (Superdex 200 10 / 300).

[0169] Figure 57 shows the experimental results of QHJ-02M02 cells.

[0170] Figure 58 shows the changes in tumor volume in mice during drug administration in each group.

[0171] Figure 59 shows the statistical graph of tumor volume in each group of mice. Detailed Implementation

[0172] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used is conventional equipment, and the equipment and materials used in each embodiment are the same. Unless otherwise specified, the materials and reagents used in the following embodiments can be obtained commercially.

[0173] Basic Implementation

[0174] Figure 1 shows a schematic diagram of the general formula for constructing the five protein aggregates involved in this invention.

[0175] The sequences and numbers of the signal peptides are shown in the table below:

[0176] Table 1

[0177] The reference compound of the present invention is N-803 (QHJ-02V02: containing IL15 (SEQ ID NO:5) and IL-15RαFc (SEQ ID NO:6).

[0178] The mature protein sequences of the five novel IL-2 / IL-15 chimeras and IL-15Ra Fc protein dimers described in this invention are as follows:

[0179] QHJ-02V03: Contains an IL-2 / IL-15 chimera (SEQ ID NO:7) and an IL-15RαFc (SEQ ID NO:6).

[0180] QHJ-02V05: Contains an IL-2 / IL-15 chimera (SEQ ID NO:8) and an IL-15RαFc (SEQ ID NO:6).

[0181] QHJ-02V13: Contains an IL-2 / IL-15 chimera (SEQ ID NO:9) and an IL-15RαFc (SEQ ID NO:6).

[0182] QHJ-02V15: Contains an IL-2 / IL-15 chimera (SEQ ID NO:9) and an IL-15RαFc (SEQ ID NO:10).

[0183] QHJ-02M02: Contains an IL-2 / IL-15 chimera (SEQ ID NO:9) and an IL-15RαFc (SEQ ID NO:11).

[0184] SEQ ID NO:5:

[0185] SEQ ID NO:6:

[0186] The DNAORF sequences corresponding to the above 5 polymers are as follows:

[0187] QHJ-02V02 includes:

[0188] (1)IL15:SEQ ID NO:18.

[0189] (2) IL-15Ra Fc: SEQ ID NO: 13.

[0190] QHJ-02V03 includes:

[0191] (1) IL-2 / IL-15 chimera: SEQ ID NO:12.

[0192] (2) IL-15Ra Fc: SEQ ID NO: 13.

[0193] QHJ-02V05 includes:

[0194] (1) IL-2 / IL-15 chimera: SEQ ID NO:14.

[0195] (2) IL-15Ra Fc: SEQ ID NO: 13.

[0196] QHJ-02V13 includes:

[0197] (1) IL-2 / IL-15 chimera: SEQ ID NO:15.

[0198] (2) IL-15Ra Fc: SEQ ID NO: 13.

[0199] QHJ-02V15 includes:

[0200] (1) IL-2 / IL-15 chimera: SEQ ID NO:15.

[0201] (2) IL-15Ra Fc: SEQ ID NO: 16.

[0202] QHJ-02M02 includes:

[0203] (1) IL-2 / IL-15 chimera: SEQ ID NO:15.

[0204] (2) IL-15Ra Fc: SEQ ID NO: 17.

[0205] SEQ ID NO:17:

[0206] SEQ ID NO:18:

[0207] Figure 2 shows the plasmid maps of IL15 and IL-15Ra Fc corresponding to the above QHJ-02V02 polymer.

[0208] The plasmid maps of the above-mentioned QHJ-02V03 polymers expressing IL-2 / IL-15 chimeras and IL-15Ra Fc are shown in Figure 3.

[0209] The plasmid maps of the above-mentioned QHJ-02V05 polymers expressing IL-2 / IL-15 chimeras and IL-15Ra Fc are shown in Figure 4.

[0210] The plasmid maps of the above-mentioned QHJ-02V13 polymers expressing IL-2 / IL-15 chimeras and IL-15Ra Fc are shown in Figure 5.

[0211] The plasmid maps of the above-mentioned QHJ-02V15 polymers expressing IL-2 / IL-15 chimeras and IL-15Ra Fc are shown in Figure 6.

[0212] The plasmid maps of the above-mentioned QHJ-02M02 polymers expressing IL-2 / IL-15 chimeras and IL-15Ra Fc are shown in Figure 7.

[0213] Example 1: Vector construction, cell culture, transient expression

[0214] 1. Plasmid construction

[0215] As shown in Figures 2-7, the DNA fragments for constructing plasmids expressing IL-15 or IL-2 / IL-15 chimeras and plasmids expressing IL-15RαFc were synthesized by GeneScript (Nanjing, China). All components of the plasmids were amplified by PCR using PrimeSTAR Max (Takara, Tokyo, Japan) and inserted into the plasmids according to the instructions of the Seamless Cloning Kit (Beyotime, China). The primer list is shown in Table 2 below. After sequencing verification, the plasmids were purified using a restriction endonuclease-free maxi-prep kit (QIGEN, Germany) and then stored at -20°C.

[0216] Table 2 Primers used for constructing plasmids for protein expression

[0217] 2. Cell culture and transient expression

[0218] (1) Expo 293F cells (Thermo Fisher Scientific) suspension culture were grown in Balance CD 293 medium (CELL-WISE) containing penicillin / streptomycin in an incubator at 37℃, 80% relative humidity and 8% CO2.

[0219] (2) Preparation of plasmids and transfection reagents: Polyethyleneimine (PEI, Alfa Aesar) was prepared by adding plasmid DNA solution to warm (37°C) Opti-MEM (Gibco), then adding PEI solution, gently mixing, and incubating at room temperature for 20 minutes. The two plasmids were co-transfected at a ratio of 1:1 or 5:1. The total plasmid amount was 100 μg / 100 mL cells.

[0220] (3) The cells were kept in a shake flask and scaled up to 0.1 L, with a seeding density of 2.8 × 10⁻⁶. 6 Cells / ml, used for transfection experiments using PEI.

[0221] (4) 20 hours after transfection, add transfection enhancer (VGlucose (400g / L):VVPA (100g / 1.5L) = 7:1) (20ml / L cells), and gently rotate the flask during the addition process;

[0222] (5) During the production period (5 days), keep the culture in a humid incubator;

[0223] (6) At 4°C, centrifuge at 2000 RPM to remove most of the cells, and then centrifuge at 8000 RPM to further remove the remaining cells and collect the protein supernatant. The supernatant is then used directly for purification.

[0224] Example 2: Detection of protein expression levels of QHJ-02V02, QHJ-02V03, and QHJ-02V05

[0225] (1) First, pre-equilibrate the protein A resin with 1×PBS (pH 7.4) at 4℃, then add 100mL of PBS.

[0226] The protein supernatant (S, supernatant) was incubated with 0.5 mL of protein A resin for 1 hour.

[0227] (2) Centrifuge at 1000 rpm for 5 minutes to collect the supernatant (FT, flow-through) and use 1 mL of 1×PBS.

[0228] Wash the resin twice (pH 7.4);

[0229] (3) Elute the target protein three times with 1 mL of elution buffer (0.1 M glycine, pH 3.0);

[0230] (4) Add 100 μL of neutralization buffer (1M Tris pH 8.0) per 1 mL of elution buffer.

[0231] Immediately adjust the elution fraction to the physiological pH.

[0232] The test results are shown in Table 3 below:

[0233] Table 3

[0234] As can be seen from the table above:

[0235] (1) Increasing the plasmid co-transfection ratio from 1:1 to 5:1 did not increase the protein expression level.

[0236] (2) The protein expression level of QHJ-02V03 is about twice that of QHJ-02V02.

[0237] (3) Comparing QHJ-02V05 with QHJ-02V03, it was found that the C-terminal labeling had little effect on protein yield.

[0238] Example 3: Gel electrophoretic protein characterization of QHJ-02V02, QHJ-02V03, and QHJ-02V05

[0239] (1) The results of SDS-PAGE 12% gel electrophoresis of QHJ-02V02 protein purified by column A are shown in Figure 8.

[0240] In the SDS-PAGE gel electrophoresis images using β-mercaptoethanol reducing agent (without PNGase F, N-glycosidase F treatment), the molecular weights of the proteins are approximately 38 KD, 16 KD, and 13 KD. The expected molecular weights of the unglycosylated protein peptide chains are 33.4 KD and 12.8 KD. The protein with a molecular weight of approximately 38 KD has a molecular weight of approximately 80 KD in the corresponding unreduced SDS-PAGE gel electrophoresis image, consistent with the expected size, which corresponds to the IL-15Rα(I1-R65)-Fc(CH2-CH3) glycosylated protein peptide chain. The two electrophoretic bands with molecular weights of approximately 16 KD and 13 KD correspond to IL15(N1-S114, N72D) glycosylated and unglycosylated protein peptide chains.

[0241] The proteins obtained after treatment with PNGase F and N-glycosidase F had molecular weights of approximately 36 KD and 13 KD, respectively. The expected molecular weights of the unglycosylated protein peptide chains were 33.4 KD and 12.8 KD, which are roughly consistent in size, corresponding to the two protein peptide chains IL-15Rα(I1-R65)-Fc(CH2-CH3) and IL15(N1-S114,N72D). The proteins obtained before treatment with PNGase F and N-glycosidase F had molecular weights of approximately 38 KD, 16 KD, and 13 KD (Figure 9).

[0242] (2) The results of SDS-PAGE 12% gel electrophoresis of QHJ-02V03 protein purified by column A are shown in Figure 10.

[0243] In the SDS-PAGE gel electrophoresis pattern using β-mercaptoethanol reducing agent (without PNGase F, N-glycosidase F treatment), the molecular weights of the proteins are approximately 38 KD, 19 KD, 15 KD, and 14 KD. The expected molecular weights of the unglycosylated protein peptide chains are 33.4 KD and 14 KD. The protein with a molecular weight of approximately 38 KD has a molecular weight of approximately 80 KD in the corresponding unreduced SDS-PAGE gel electrophoresis pattern, consistent with the expected size, which corresponds to the IL-15Rα(I1-R65)-Fc(CH2-CH3) glycosylated protein peptide chain. The three electrophoretic bands with molecular weights of approximately 19 KD, 15 KD, and 14 KD correspond to IL-15 (N72D) highly glycosylated, moderately glycosylated, and low / unglycosylated protein peptide chains.

[0244] The proteins treated with PNGase F and N-glycosidase F have molecular weights of approximately 36 KD, 16 KD, and 14 KD, respectively. 36 KD corresponds to the IL-15Rα(I1-R65)-Fc(CH2-CH3) protein peptide chain, 14 KD corresponds to the unglycosylated or deglycosylated IL-2 / IL-15 chimeric (N72D) protein peptide chain, and 16 KD corresponds to the incompletely deglycosylated IL-2 / IL-15 chimeric (N72D) protein peptide chain. Because N-glycosidase F can only cleave completely exposed N-linked glycosyl groups on the protein surface and cannot cleave O-linked glycosyl groups, the QHJ-02V03 protein is more severely glycosylated than the QHJ-02V02 protein (Figure 11).

[0245] (3) The results of SDS-PAGE 12% gel electrophoresis of QHJ-02V05 protein purified by column A are shown in Figure 12.

[0246] In the SDS-PAGE gel electrophoresis pattern using β-mercaptoethanol reducing agent, there is a thick band with a molecular weight of approximately 38 kDa and a cluster of bands in the 15 kDa and 20 kDa regions. The expected molecular weights of the unglycosylated protein peptides are 33.4 kDa and 15 kDa. Proteins with a molecular weight of approximately 38 kDa have a molecular weight of approximately 80 kDa in the corresponding unreduced SDS-PAGE gel electrophoresis pattern, which corresponds to the expected IL-15Rα(I1-R65)-Fc(CH2-CH3) protein peptides. The electrophoretic bands with molecular weights between 20 kDa and 15 kDa correspond to various IL-2 / IL-15 chimeric (N72D) glycosylated and low / unglycosylated protein peptides.

[0247] The N-terminus of the IL-2 / IL-15 chimeric protein chain corresponding to QHJ-02V05 also contains the APTSSSTKKTQL polypeptide. Similar to QHJ-02V03, its molecular weight is also significantly higher than expected, reflecting severe glycosylation on the corresponding IL15 protein chain.

[0248] Example 4: Further purification and characterization of QHJ-02V02 and QHJ-02V03 proteins

[0249] Further purification of QHJ-02V02 and QHJ-02V03 proteins was performed according to the steps in the literature (Han, KP, Zhu, X., Liu, B., Jeng, E., Kong, L., & Yovandich, JL, et al. (2011). IL-15: IL-15 receptor alpha superagonist complex: high-level co-expression in recombinant mammalian cells, purification and characterization. Cytokine, 56(3), 804-810.). 0.6 L of the protein expressed by Expi293F was passed through a protein A column, the eluted portion was passed through a QHP column, and then purified through a SEC column. The QHP column buffer used was:

[0250] Buffer A: 20 mM Tris, pH 8.0;

[0251] Buffer B: 20mM Tris, 1M NaCl, pH 8.0;

[0252] The SEC column buffer used was PBS, pH 7.4.

[0253] (1)QHJ-02V02

[0254] Purified by QHP (5 ml) column (Figure 13) and detected by SDS-PAGE (Figure 14).

[0255] Purified via SEC column (Superdex 200 10 / 300) (Figures 15-18).

[0256] The SDS-PAGE purity analysis results are shown in Figure 19.

[0257] SEC Analysis Column Test:

[0258] Flow rate: 0.4 ml / min;

[0259] aSEC Buffer:PBS, pH 7.4;

[0260] aSEC Column:Superdex 200Increase 5 / 150GL.

[0261] The results for peak #1 are shown in Figure 20. Retention time: 3.658 min. The calculated apparent molecular weight (assuming globulin shape) is 122.4 KD.

[0262] The results for peak #2 are shown in Figure 21. Retention time: 3.638 min. The calculated apparent molecular weight (assuming globulin shape) is 126.7 KD.

[0263] Natural intact protein profile analysis: The results of peak #1 are shown in Figure 22. Among the main peaks detected in the sample with peak #1, there are unglycosylated IL-15-containing protein peptide chains (molecular weight 12.772 KD), glycosylated IL-15-containing protein peptide chains (molecular weight between 14.833 KD and 15.489 KD, with a maximum peak at 14.833 KD), and glycosylated IL-15Ra Fc fusion protein peptide chains (molecular weight between 69.459 KD and 69.946 KD, with a maximum peak at 69.459 KD).

[0264] The results for peak #2 are shown in Figure 23. Among the main peaks detected in the sample of peak #2, there are unglycosylated IL-15-containing protein peptide chains (molecular weight 12.772 KD), glycosylated IL-15-containing protein peptide chains (molecular weight between 14.833 KD and 15.489 KD, with a maximum peak of 15.124 KD), and glycosylated IL-15Ra Fc fusion protein peptide chains (molecular weight between 69.459 KD and 69.946 KD, with a maximum peak of 69.459 KD).

[0265] Therefore, it can be seen that, regardless of whether it is peak #1 or peak #2, the main peak detected in the IL-15 protein peptide chain of the QHJ-02V02 molecule is the glycosylated protein peptide chain.

[0266] (2)QHJ-02V03

[0267] Purified by QHP (5 ml) column (Figure 24) and detected by SDS-PAGE (Figure 25).

[0268] Purified via SEC column (Superdex 200 10 / 300) (Figures 26-29).

[0269] The SDS-PAGE purity analysis is shown in Figure 30.

[0270] SEC Analysis Column Test:

[0271] Flow rate: 0.4 ml / min;

[0272] aSEC Buffer:PBS, pH 7.4;

[0273] aSEC Column:Superdex 200Increase 5 / 150GL.

[0274] The results for peak #1 are shown in Figure 31. Retention time: 3.591 min. The calculated apparent molecular weight (assuming globulin shape) is 137.3 KD.

[0275] The results for peak #2 are shown in Figure 32. Retention time: 3.535 min. The calculated apparent molecular weight (assuming globulin shape) is 151.0 KD.

[0276] Natural intact protein proteomic analysis:

[0277] The results for peak #1 are shown in Figure 33. In the IL-2 / IL-15-containing protein peptide chains of the sample with peak #1, the main peak detected was a glycosylated protein peptide chain (molecular weight of 14.95 KD), and no unglycosylated IL-2 / IL-15-containing protein peptide chains were detected. The mass distribution of the IL-15Ra Fc fusion protein peptide chain was basically consistent with that of the QHJ-02V02 sample (the molecular weight of the IL-15Ra Fc homodimer was between 69.459 KD and 69.946 KD, with the maximum peak at 69.459 KD).

[0278] The results for peak #2 are shown in Figure 34. In the IL-2 / IL-15-containing protein peptide chains of the sample with peak #2, the main peak detected was also a glycosylated protein peptide chain (molecular weight of 14.95 KD), and no unglycosylated IL-2 / IL-15-containing protein peptide chains were detected. The mass distribution of the IL-15Ra Fc fusion protein peptide chains was also basically consistent with that of the QHJ-02V02 sample (the molecular weight of the IL-15Ra Fc homodimer was between 69.459 KD and 69.946 KD, with the maximum peak at 69.459 KD).

[0279] Conclusion: The results of SDS-PAGE gel electrophoresis (including PNGase F glycosidase treatment), SEC analysis, and native intact proteomic analysis all indicate that the IL-2 / IL-15-containing peptide chains in QHJ-02V03 have a higher molecular weight than those in QHJ-02V02 and have additional glycosylation.

[0280] Example 5: Evaluation of the stimulatory effects of QHJ-02V02 and QHJ-02V03 on cell proliferation in the Mo7e cell line

[0281] 1. Main materials and reagents

[0282] 1.1 RPMI-1640 (Gibco, part number 22400089);

[0283] 1.2 FBS (Gibco, part number 10091130);

[0284] 1.3 Chemiluminescence cell viability assay (Promega, Cat. No. G7572, 100 ml);

[0285] 1.4 96-well tissue culture plate (Grenier, Cat. No. 655180);

[0286] 1.5 human IL-15 protein, premium grade (Acro, Cat. No. IL5-H5215);

[0287] 1.6 Cell line: M07e;

[0288] 1.7 Protein concentration assay method: Bradford; Protein sample storage buffer: PBS, pH 7.4.

[0289] 2. Methods

[0290] 2.1 Experimental Procedure

[0291] 1) To measure cytokine-dependent cell proliferation, M-07e cells were harvested during the logarithmic growth phase and washed twice with PBS.

[0292] 2) Cells were then seeded into 96-well cell culture plates at a density of 20,000 cells / 90 μL / well. The plates were incubated at 37°C in a humid atmosphere with 5% CO2 for 2 hours.

[0293] 3) Two hours later, treat cells with different concentrations of IL-15 or its analogues or other proteins diluted with PBS (12-point curve, starting from 100 nM, 3× dilution, triplicate). Add 10 μl to each well of the cell culture system. Add the highest concentration of IL-15 as a positive control (max), and add 10 μl / well of PBS as a negative control (min). Incubate the cells at 37°C for 72 hours.

[0294] 4) On day 5, add Cell Titer-Glo reagent to each well and centrifuge at 1000 rpm for 30 seconds. Incubate with shaking at RT for 15-30 minutes.

[0295] 5) Read the board on the Envision (Perkin Elmer) board reader.

[0296] 6) PBS, as a negative control, showed the same level of effect as the blank signal and did not stimulate cell growth and proliferation.

[0297] 3. Data Analysis

[0298] Use GraphPad Prism 8.0 software to process and analyze data.

[0299] Experimental sample:

[0300] Table 4

[0301] *The theoretical molecular weight (KD) here refers to the total molecular weight of amino acids contained in a single IL-5 molecule or a single IL-2 / IL-15 chimeric protein and IL-15Ra Fc complex, not the molecular weight of the complex dimer, and the molecular weight calculation does not include glycosylated groups.

[0302] Cellular experiment results:

[0303] (1) The results of the IL-15 (Acro, #IL5-H5215) cell experiment are shown in Figure 35.

[0304] (2) The results of the QHJ-02V02 (#1 peak) cell experiment are shown in Figure 36.

[0305] (3) The results of the QHJ-02V02 (#2 peak) cell experiment are shown in Figure 37.

[0306] (4) The results of the QHJ-02V03 (#1 peak) cell experiment are shown in Figure 38.

[0307] (5) The results of the QHJ-02V03 (#2 peak) cell experiment are shown in Figure 39.

[0308] in conclusion:

[0309] (1) Both QHJ-02V02 and QHJ-02V03 have a strong proliferative effect on Mo7e cells.

[0310] (2) Compared with IL-15, QHJ-02V02 and QHJ-02V03 can increase the half-maximum proliferation value (EC50) of Mo7e cell lines by 3 to 6 times, and the EC50 difference between QHJ-02V02 and QHJ-02V03 is not significant.

[0311] (3) The cell activities of peak #1 and peak #2 of QHJ-02V02 were similar, while the cell activities of peak #1 and peak #2 of QHJ-02V03 were not significantly different.

[0312] Example 6: In vivo study of tumor inhibition by QHJ-02V02 in a mouse B16F10 melanoma model

[0313] 1. Experimental Materials

[0314] 1.1 Test Sample 1

[0315] Table 5

[0316] Appearance: A colorless or nearly colorless clear isotonic liquid.

[0317] 1.2 Test Sample 2

[0318] Human IL-15 Protein# (HEK293), purchased from Acro Biosystems, catalog number IL5-H5215, appearance: white powder.

[0319] 2. Experimental animals

[0320] Six-week-old female C57BL / 6N mice were purchased from Shanghai Shengchang Biotechnology Co., Ltd. The experimental animals were housed in an SPF barrier environment at Shanghai Shengchang Biotechnology Co., Ltd., using individually ventilated (IVC) cages. Before the experiment, the animals underwent 3-7 days of acclimatization. The housing conditions were: temperature 22±2℃, humidity 40-70%, and a 12h / 12h diurnal cycle.

[0321] Mice were fed a normal breeding diet (sterilized and sealed packaging) purchased from Shanghai Proton Biotechnology Co., Ltd. Drinking water was self-prepared purified water that had undergone sterilization. Animals had free access to sterile food and water.

[0322] 3. Experimental reagents and instruments

[0323] The electronic balance was purchased from Shanghai Jingtian Electronic Instruments Co., Ltd., item number JT201N; the disposable syringe was purchased from Shanghai Kangdelai Enterprise Development Group Co., Ltd., item number K20190705.

[0324] 4 Experimental Methods

[0325] 4.1 Experimental Procedure

[0326] 4.1.1 Drug Preparation

[0327] Preparation of IL15-2μg: ​​Dissolve 20μg of the drug in 2ml of 1xPBS.

[0328] Preparation of the required reagents for QHJ-02V02p1-5μg:

[0329] Dissolve 0.05 mg of QHJ-02V02p1 in 2 ml of 1xPBS.

[0330] Preparation of the required reagents for QHJ-02V02p1-15μg:

[0331] Dissolve 0.15 mg of QHJ-02V02p1 in 2 ml of 1xPBS.

[0332] Preparation of the required reagents for QHJ-02V02p2-5μg:

[0333] Dissolve 0.05 mg of QHJ-02V02p2 in 2 ml of 1xPBS.

[0334] Preparation of the required reagents for QHJ-02V02p2-15μg:

[0335] Dissolve 0.15 mg of QHJ-02V02p2 in 2 ml of 1xPBS.

[0336] 4.2 Grouping and Detection Indicators of Experimental Animals

[0337] Table 6

[0338] Each mouse was injected with 5*10 6 B16F10 cells were inoculated, and tumor size was measured on day 7 post-inoculation. Mice were randomly divided into 6 groups (excluding 2 tumor-bearing mice with abnormal tumors), with 8 tumor-bearing mice in each group: Group 1 (1xPBS, 200μL, IP), Group 2 (IL15-2μg, 200μL, IP), Group 3 (QHJ-02V02p1-5μg, 200μL, IP), Group 4 (QHJ-02V02p1-15μg, 200μL, IP), Group 5 (QHJ-02V02p2-5μg, 200μL, IP), and Group 6 (QHJ-02V02p2-15μg, 200μL, IP). The theoretical molecular weight of IL15 is 12.8 kDa; therefore, the molar concentration corresponding to 2 μg / 200μL is 0.78 μM. The theoretical molecular weight of QHJ-02V02 heterodimer (containing a single IL15) is 46.2 kDa. Therefore, the molar concentrations corresponding to 5 μg / 200 μL and 15 μg / 200 μL are 0.54 μM and 1.62 μM, respectively.

[0339] The first day after grouping the mice is designated as day1, and the first day after grouping is designated as day2 (first administration). The mice are administered the drug on day2 and day6, and the changes in tumor volume are recorded.

[0340] Testing indicators:

[0341] (1) Tumor volume:

[0342] Calculation formula: V = 1 / 2 × L length ×L short 2 .

[0343] 5. Data Analysis

[0344] Results are expressed as mean ± standard error and analyzed using Prism or SPSS. A p-value < 0.05 is considered statistically significant.

[0345] 6. Experimental Results

[0346] 6.1 Tumor Measurement

[0347] The changes in tumor volume during drug administration are shown in Figure 40, and the tumor volume statistics are shown in Figure 41.

[0348] The statistical values ​​between each pair are shown in the table below:

[0349] Table 7

[0350] The Mean values ​​of tumor volume in Group 1-Group 6 mice were 4395.880396, 2817.337735, 2419.31742, 1283.106319, 2267.430664, and 1007.591431 mm, respectively. 3 Regarding tumor size, the tumors in Groups 2-6 were smaller compared to those in Group 1 (PBS). The high-dose Group 4 (QHJ-02V02p1-15μg) and Group 6 (QHJ-02V02p2-15μg) groups showed a greater decreasing trend in tumor volume than the low-dose Group 3 (QHJ-02V02p1-5μg) and Group 5 (QHJ-02V02p2-5μg) groups. The tumor volume in Groups 4 (QHJ-02V02p1-15μg) and Group 6 (QHJ-02V02p2-15μg) was significantly lower than that in Group 2 (IL15-2μg). There was no statistically significant difference between Group 3 (QHJ-02V02p1-5μg) and Group 5 (QHJ-02V02p2-5μg), and similarly, there was no statistically significant difference between Group 4 (QHJ-02V02p1-15μg) and Group 6 (QHJ-02V02p2-15μg).

[0351] In summary, all treatment groups showed therapeutic effects on subcutaneous melanoma in C57 mice. The efficacy of QHJ-02V02p2-15μg and QHJ-02V02p2-15μg was superior to that of the IL15-2μg group, but there were no statistically significant differences in efficacy among the other treatment groups. There was no statistically significant difference in efficacy between QHJ-02V02p1 and QHJ-02V02p2.

[0352] Compared with the PBS group, QHJ-02V02p1 and QHJ-02V02p2 could effectively reduce the tumor volume and tumor area, and compared with the Group 2 (IL15-2μg) group, the tumor volume and tumor area were significantly reduced, indicating that QHJ-02V02p1 and QHJ-02V02p2 could effectively treat subcutaneous melanoma.

[0353] Example 7: In vivo study on the inhibition of lung metastasis of mouse B16F10 melanoma model

[0354] 1. Experimental purpose

[0355] The purpose of this experiment was to study the growth inhibitory effect of the test drug on animals in the B16F10 melanoma lung metastasis model.

[0356] 2. Experimental materials

[0357] 2.1 Tumor cells

[0358] B16F10 mouse melanoma cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. The cells were passaged once every two days and placed in an incubator at 37°C and 5% CO2 for culture. The tumor cells in the logarithmic growth phase were transplanted in vivo and continued to be cultured in an incubator at 37°C and 5% CO2. The tumor cells in the logarithmic growth phase were used to establish an in vivo transplanted tumor model.

[0359] 2.2 Experimental animals

[0360] 45 C57BL / 6N mice, female, 7-8 weeks old, weighing about 19g-21g. Purchase date: April 9, 2024.

[0361] The experimental animals were obtained from Vital River Laboratory Animal Technology Co., Ltd., certificate number: 20240409Abzz0619000169.

[0362] The production license number of experimental animals: SCXK(Zhe)2024-0001.

[0363] The use license number of experimental animals: SYXK(Shanghai)-2021-0021 (Lijin Biopharmaceutical Technology (Shanghai) Co., Ltd.).

[0364] 2.2.1 Feeding of experimental animals

[0365] All mice were housed in an SPF-grade animal facility with an IVC (Indoor Temperature and Pressure) system, maintaining a temperature of 20-26°C, humidity of 40-70%, and a 12-hour light-dark cycle. Each cage contained 3-5 mice and measured 325mm x 210mm x 180mm. Bedding was changed twice weekly. Throughout the experiment, all mice had free access to food and water, which were autoclaved and changed twice weekly. All personnel entering and leaving the animal housing or performing experimental procedures wore disposable sterile lab coats, disposable medical masks, and rubber gloves. Each cage had a clear and detailed label including: number of animals, sex, strain, date of receipt, project number, group, current experimental stage, and person in charge. Animals were numbered using ear punches.

[0366] 2.3 Test substance

[0367] Table 8

[0368] 2.4 Other reagents and experimental instruments

[0369] Table 9

[0370] 3. Experimental Procedures and Methods

[0371] 3.1 Cell Seeding

[0372] B16F10 cells resuspended in PBS were charged at a dose of 1×10⁻⁶. 5 A 0.2 mL dose was administered to mice via tail vein injection. A total of 42+3 (reserve) mice were transfected, with each mouse receiving 0.2 mL (containing 1 × 10⁵ cells). Administration began on the day of inoculation, with three doses administered on days 1, 2, and 10, for a total of three administrations. Administration began on the day of grouping. The specific administration regimen is shown in the table below:

[0373] Table 10. Route of administration, dosage, and grouping for the pharmacodynamic experiment.

[0374] The theoretical molecular weight of the QHJ-02V03 heterodimer (containing a single IL15) is 47.4 kDa. Therefore, the molar concentrations corresponding to 5 μg / 0.2 mL and 15 μg / 0.2 mL are 0.53 μM and 1.58 μM, respectively.

[0375] 3.2 Preparation of test substance administration solution

[0376] The preparation of the test substance administration solution is shown in the table below:

[0377] Table 11 Preparation of Drug Solution

[0378] 3.3 Experimental Observation

[0379] Throughout the experiment, the use and observation of laboratory animals were conducted in accordance with the relevant regulations for the use and management of animals by AAALAC. After inoculation with tumor tissue, the laboratory animals were observed daily, and their morbidity and mortality were recorded. In accordance with standard experimental procedures, all laboratory animals were monitored and recorded for behavior, food and water intake, weight changes, coat luster, and other abnormalities.

[0380] 3.4 Evaluation Indicators

[0381] Weight monitoring: BWL(%) = (BWi-BW0) / BW0×100, where BWi is the average weight of mice in the group on a certain day after grouping, and BW0 is the average weight of mice on the first day when treatment begins.

[0382] Body temperature monitoring: One week after modeling, the body temperature of the animals was measured and recorded at fixed times every day. The body temperature of each group of animals was observed and recorded during days 10-16. If the body temperature was found to be below 33℃, the animals were euthanized in time and specimens were collected, or the client was contacted to discuss whether to end the experiment.

[0383] Counting lung metastases: Euthanize the animal, dissect and remove the lung, rinse and fix it with formalin solution, and count the black spots (tumors) in the lung. It is necessary to count both the front and back of the lung at the same time.

[0384] 3.5 Experiment Termination

[0385] The experiment was conducted with three doses of medication, ending on day 16. Animals were euthanized, lung tissue was completely dissected, and lung tumor lesions were photographed and counted. The lung tissue was then preserved in formalin.

[0386] 3.6 Data Analysis

[0387] All data are expressed as mean ± SEM. SPSS 23.0 statistical analysis software was used to perform normality and homogeneity of variance tests. One-way ANOVA was selected based on the test results. *P < 0.05 was considered statistically significant.

[0388] 4. Experimental Results

[0389] 4.1 Weight monitoring

[0390] B16F10 cells resuspended in PBS were charged at a dose of 1×10⁻⁶. 5The drug was administered at a concentration of 0.2 mL via tail vein injection to mice, who were then randomly divided into 6 groups (7 mice per group). The drugs were also administered intraperitoneally on the same day. The experiment was concluded after 16 days of observation. The body weight curves for each treatment group and the control group during the experiment are shown in Table 12 and Figure 42 below.

[0391] Table 12. Mean body weight change data of tumor-bearing mice during drug administration (Mean±SEM, n=7)

[0392] 4.2 Body temperature monitoring

[0393] The weight curves of each treatment group and the control group during the experiment are shown in Table 13 and Figure 43.

[0394] Table 13. Mean body temperature changes in tumor-bearing mice during drug administration (Mean±SEM, n=7)

[0395] 4.3 Count of lung metastases

[0396] At the experimental endpoint (Day 16), the lung metastasis status of tumor-bearing mice in each group is shown in Table 14, Figure 44, and Figures 45-50 below.

[0397] Table 14 Inhibitory effect of the test substance on the B16F10 lung metastasis model (n=7) Note: a : This represents the tumor metastasis inhibition rate, calculated using the formula: (%) = (1 - TW) treatment / TW vehicle )×100%, of which TW treatment TW represents the average number of metastatic lesions in the treatment group at the experimental endpoint. vehile This represents the average number of metastatic lesions in the negative control group at the endpoint of the experiment.

[0398] p-value 1 The results of the statistical analysis show the number of lung metastases in each treatment group (G2-G6) and the number of lung metastases in the control group (G1).

[0399] p-value 2 : The statistical analysis results of the number of lung metastases in the treatment groups G3-G6 and the number of lung metastases in the positive control group (G2).

[0400] 5. Experiment Summary

[0401] This experiment tested the antitumor effects of different doses of QHJ-02V02 and QHJ-02V03 in a B16F10 melanoma lung metastasis model. Six groups of female BALB / c nu tumor-bearing mice (n=7 per group) were included in the experiment. Each group was administered a blank solvent, different doses of QHJ-02V02 and QHJ-02V03, and IL-15 as a positive control, respectively, via intraperitoneal injection.

[0402] At the end of the experiment (day 16), the average number of lung metastases in the solvent control group G1 was 45.86, with a metastasis rate of 100%, indicating that the B16F10 lung metastasis modeling was successful.

[0403] At the end of treatment (day 16), the metastasis rates of the G3 group (using QHJ-02V02 5ug / animal), the G4 group (using QHJ-02V02 15ug / animal), and the G5 and G6 groups (using QHJ-02V03 5ug / animal and QHJ-02V03 15ug / animal) were 42.86%, 28.57%, 0.00%, and 28.57%, respectively. Compared with the control group G1, the lung metastasis inhibition rates were 97.51%, 99.37%, 100%, and 99.06%, respectively, and the differences were statistically significant (P < 0.05), showing a significant inhibitory effect on tumor metastasis. Among them, the G5 group treated with QHJ-02V03 5ug / animal and the G2 group (IL-15) Compared with the 90.03% inhibition rate of metastatic lesions (2ug / animal), the inhibitory effect on metastasis was significantly improved, and the difference was statistically significant (P<0.05).

[0404] In summary, different doses of QHJ-02V02 and QHJ-02V03 demonstrated significant anti-tumor metastasis effects in a B16F10 melanoma lung metastasis model animal; no significant dose-dependent relationship was observed between the two tested drugs at treatment doses of 5 μg / animal and 15 μg / animal. Furthermore, QHJ-02M01 and QHJ-02M02 significantly inhibited tumor metastasis.

[0405] In summary, the novel IL-15 agonist of the present invention includes an IL-15 analog protein and an IL-15Rα analog protein. The IL-15 analog protein has a larger molecular weight than the IL-15 protein and more glycosylations on the protein. The IL-15Rα analog protein includes an IL-15RαSushi domain and an IgG1 Fc CH2-CH3 domain.

[0406] QHJ-02V03 showed higher protein expression and glycosylation levels than QHJ-02V02 (N-803), exhibiting significant anti-tumor metastasis effects in the B16F10 melanoma lung metastasis model and a significant therapeutic effect on subcutaneous melanoma in C57 mice.

[0407] Example 8: Purification and characterization of QHJ-02V13, QHJ-02V15, and QHJ-02M02

[0408] The general purification steps for QHJ-02V13, QHJ-02V15, and QHJ-02M02 are as follows:

[0409] (1) First, pre-equilibrate the protein A resin with 1×PBS (pH 7.4) at 4°C, and then incubate 100 mL of protein supernatant (S, supernatant) with 0.5 mL of protein A resin for 1 hour.

[0410] (2) Centrifuge at 1000 rpm for 5 minutes to collect the supernatant (FT, flow through), and wash the resin twice with 1 mL of 1×PBS (pH 7.4);

[0411] (3) Elute the target protein three times with 1 mL of elution buffer (0.1 M glycine, pH 3.0);

[0412] (4) Adjust the elution fraction to physiological pH immediately by adding 100 μL of neutralization buffer (1M Tris pH 8.0) per 1 mL of elution fraction.

[0413] (5) The eluent after protein A purification was concentrated and purified further using a SEC column (Superdex 200 10 / 300). The SEC column buffer used was PBS (pH 7.4).

[0414] Figure 51 shows the SDS-PAGE 12% gel electrophoresis results of the QHJ-02V13 protein purified by column A, and Figure 52 shows the results after further purification by column SEC.

[0415] Figure 53 shows the SDS-PAGE 12% gel electrophoresis results of the QHJ-02V15 protein purified by column A, and Figure 54 shows the results after further purification by column SEC.

[0416] Figure 55 shows the SDS-PAGE 12% gel electrophoresis results of the QHJ-02M02 protein purified by column A, and Figure 56 shows the results after further purification by column SEC.

[0417] Example 9: Evaluation of the stimulatory effect of QHJ-02M02 on cell proliferation in the Mo7e cell line

[0418] 1. Main materials and reagents

[0419] 1.1 RPMI-1640 (Gibco, part number 22400089);

[0420] 1.2 FBS (Gibco, part number 10091130);

[0421] 1.3 Chemiluminescence cell viability assay (Promega, Cat. No. G7572, 100 ml);

[0422] 1.4 96-well tissue culture plate (Grenier, Cat. No. 655180);

[0423] 1.5 human IL-15 protein, premium grade (Acro, Cat. No. IL5-H5215);

[0424] 1.6 Cell line: M07e;

[0425] 1.7 Protein concentration assay method: Bradford; Protein sample storage buffer: PBS, pH 7.4.

[0426] 2. Methods

[0427] 2.1 Experimental Procedure

[0428] 1) To measure cytokine-dependent cell proliferation, M-07e cells were harvested during the logarithmic growth phase and washed twice with PBS.

[0429] 2) Cells were then seeded into 96-well cell culture plates at a density of 20,000 cells / 90 μL / well. The plates were incubated at 37°C in a humid atmosphere with 5% CO2 for 2 hours.

[0430] 3) Two hours later, treat cells with different concentrations of IL-15 or its analogues or other proteins diluted with PBS (12-point curve, starting from 100 nM, 3× dilution, triplicate). Add 10 μl to each well of the cell culture system. Add the highest concentration of IL-15 as a positive control (max), and add 10 μl / well of PBS as a negative control (min). Incubate the cells at 37°C for 72 hours.

[0431] 4) On day 5, add Cell Titer-Glo reagent to each well and centrifuge at 1000 rpm for 30 seconds. Incubate with shaking at RT for 15-30 minutes.

[0432] 5) Read the board on the Envision (Perkin Elmer) board reader.

[0433] 6) PBS, as a negative control, showed the same level of effect as the blank signal and did not stimulate cell growth and proliferation.

[0434] 3. Data Analysis

[0435] Use GraphPad Prism 8.0 software to process and analyze data.

[0436] Experimental sample:

[0437] Table 15 *The theoretical molecular weight (KD) here refers to the total molecular weight of amino acids contained in a single IL-5 molecule or a single IL-2 / IL-15 chimeric protein and IL-15Ra Fc complex, not the molecular weight of the complex dimer, and the molecular weight calculation does not include glycosylated groups.

[0438] Conclusion: As shown in Figure 57, QHJ-02M02 has a proliferative effect on the Mo7e cell line. The activity (EC50) of QHJ-02M02-1 (Frac2-4) is similar to that of IL-15, while the activity of QHJ-02M02-2 (Frac5-7) is weaker than that of QHJ-02M02-1 (Frac2-4).

[0439] Example 10: In vivo study of tumor inhibition by QHJ-02V03, QHJ-02V13 and QHJ-02V15 in a mouse B16F10 melanoma model

[0440] 1. Experimental Materials

[0441] 1.1 Test Sample 1

[0442] Table 16

[0443] Appearance: A colorless or nearly colorless clear isotonic liquid.

[0444] 1.2 Test Sample 2

[0445] Human IL-15 Protein# (HEK293), purchased from Acro Biosystems, catalog number IL5-H5215, appearance: white powder.

[0446] 2. Experimental animals

[0447] Six-week-old female C57BL / 6N mice were purchased from Shanghai Shengchang Biotechnology Co., Ltd. The experimental animals were housed in an SPF barrier environment at Shanghai Shengchang Biotechnology Co., Ltd., using individually ventilated (IVC) cages. Before the experiment, the animals underwent 3-7 days of acclimatization. The housing conditions were: temperature 22±2℃, humidity 40-70%, and a 12h / 12h diurnal cycle.

[0448] Mice were fed a normal breeding diet (sterilized and sealed packaging) purchased from Shanghai Proton Biotechnology Co., Ltd. Drinking water was self-prepared purified water that had undergone sterilization. Animals had free access to sterile food and water.

[0449] 3. Experimental reagents and instruments

[0450] The electronic balance was purchased from Shanghai Jingtian Electronic Instruments Co., Ltd., item number JT201N; the disposable syringe was purchased from Shanghai Kangdelai Enterprise Development Group Co., Ltd., item number K20190705.

[0451] 4 Experimental Methods

[0452] 4.1 Experimental Procedure

[0453] 4.1.1 Drug Preparation

[0454] Preparation of the required reagents for QHJ-02V03-2μg:

[0455] Dissolve 0.02 mg of QHJ-02V03 in 2 ml of 1xPBS.

[0456] Preparation of the required reagents for QHJ-02V03-5μg:

[0457] Dissolve 0.05 mg of QHJ-02V03 in 2 ml of 1xPBS.

[0458] Preparation of the required reagents for QHJ-02V13-2μg:

[0459] Dissolve 0.02 mg of QHJ-02V13 in 2 ml of 1xPBS.

[0460] Preparation of the required reagents for QHJ-02V13-5μg:

[0461] Dissolve 0.05 mg of QHJ-02V13 in 2 ml of 1xPBS.

[0462] Preparation of the required reagents for QHJ-02V15-2μg:

[0463] Dissolve 0.02 mg of QHJ-02V15 in 2 ml of 1xPBS.

[0464] Preparation of the required reagents for QHJ-02V15-5μg:

[0465] Dissolve 0.05 mg of QHJ-02V15 in 2 ml of 1xPBS.

[0466] 4.2 Grouping and Detection Indicators of Experimental Animals

[0467] Table 17

[0468] Each mouse was injected with 5*10 5 B16F10 cells were inoculated, and tumor size was measured on day 10 post-inoculation. Mice were randomly divided into 8 groups (excluding 4 tumor-bearing mice with abnormal tumors). Each group consisted of 7 tumor-bearing mice: Group 1 (1xPBS, 200μL, IP), Group 2 (IL15-1.6μg, 200μL, IP), Group 3 (QHJ-02V03-2μg, 200μL, IP), Group 4 (QHJ-02V03-5μg, 200μL, IP), Group 5 (QHJ-02V13-2μg, 200μL, IP), Group 6 (QHJ-02V13-5μg, 200μL, IP), Group 7 (QHJ-02V15-2μg, 200μL, IP), and Group 8 (QHJ-02V15-5μg, 200μL, IP).

[0469] The theoretical molecular weight of IL15 is 12.8 kDa; therefore, the molar concentration corresponding to 1.6 μg / 200 μL is 0.63 μM. The theoretical molecular weight of the QHJ-02V03 heterodimer (containing a single IL15) is 47.4 kDa; therefore, the molar concentrations corresponding to 2 μg / 200 μL and 5 μg / 200 μL are 0.21 μM and 0.53 μM, respectively. The theoretical molecular weight of the QHJ-02V13 heterodimer (containing a single IL15) is 47.4 kDa; therefore, the molar concentrations corresponding to 2 μg / 200 μL and 5 μg / 200 μL are 0.21 μM and 0.53 μM, respectively. The theoretical molecular weight of QHJ-02V15 heterodimer (containing a single IL15) is 47.5 kDa. Therefore, the molar concentrations corresponding to 2 μg / 200 μL and 5 μg / 200 μL are 0.21 μM and 0.53 μM, respectively.

[0470] The first day after grouping the mice was designated as day 0. The mice were administered the drug on day 2 and day 6, and the changes in tumor volume were recorded.

[0471] Testing indicators:

[0472] (1) Tumor volume:

[0473] Calculation formula: V = 1 / 2 × L length ×L short 2 .

[0474] 5. Data Analysis

[0475] Results are expressed as mean ± standard error and analyzed using Prism or SPSS. A p-value < 0.05 is considered statistically significant.

[0476] 6. Experimental Results

[0477] 6.1 Tumor Measurement

[0478] The changes in tumor volume during drug administration are shown in Figure 58, and the tumor volume statistics are shown in Figure 59.

[0479] The statistical values ​​between each pair are shown in Table 18 below:

[0480] Table 18

[0481] At the time of sampling, the Mean values ​​of tumor volume in mice in the PBS group, QHJ-02V03-2μg, QHJ-02V03-5μg, QHJ-02V13-2μg, QHJ-02V13-5μg, QHJ-02V15-2μg, QHJ-02V15-5μg, and IL-15 group were 3101.8, 2254.8, 1221.0, 2399.6, and 1162.3, respectively. The tumor volumes were 1606.2, 705.9, and 1702.7 mm³. In terms of tumor volume, the tumors in the drug-treated mice were slightly smaller than those in the PBS group (1xPBS, 200 μL, IP). The high-dose drug group showed a significant decrease in tumor volume compared to other treatment groups, with statistically significant differences between the drug group and the PBS group (P<0.05), especially in the QHJ-02V15-5 μg group. The QHJ-02V03-5 μg and QHJ-02V13-5 μg groups showed a decreasing trend in tumor volume compared to the low-dose QHJ-02V03-2 μg and QHJ-02V13-2 μg groups, but these differences were not statistically significant. There was no statistically significant difference in tumor volume between the QHJ-02V15-5 μg group and the low-dose QHJ-02V15-2 μg group.

[0482] [Amended according to Rule 26, 30.09.2025] The above description is only an embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. 1 A novel dimer fusion protein, characterized in that, The dimer fusion protein includes an IL-2 / IL-15 protein chimera and an IL-15RαFc fusion protein. The IL-2 / IL-15 protein chimera includes a polypeptide segment at the N-terminus of IL-2 and the full-length peptide chain of IL-15 or a variant thereof. The IL-15RαFc fusion protein includes an IL-15RαSushi domain and an Fc CH2-CH3 domain. The dimer fusion protein is: (1) QHJ-02V03: The IL-2 / IL-15 protein chimera has the sequence shown in SEQ ID NO:7, and the IL-15RαFc fusion protein has the sequence shown in SEQ ID NO:6; or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7 or 6; or; (2) QHJ-02V05: The IL-2 / IL-15 protein chimera has the sequence shown in SEQ ID NO:8, and the IL-15RαFc fusion protein has the sequence shown in SEQ ID NO:6; or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:8 or 6; or; (3) QHJ-02V13: The IL-2 / IL-15 protein chimera has the sequence shown in SEQ ID NO:9, and the IL-15RαFc fusion protein has the sequence shown in SEQ ID NO:6; or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:9 or 6; or; (4) QHJ-02V15: The IL-2 / IL-15 protein chimera has the sequence shown in SEQ ID NO:9, and the IL-15RαFc fusion protein has the sequence shown in SEQ ID NO:10; or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:9 or 10; (5)QHJ-02M02: The IL-2 / IL-15 protein chimera has the sequence shown in SEQ ID NO:9, and the IL-15RαFc fusion protein has the sequence shown in SEQ ID NO:11; or an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:9 or 11.

2. The method for preparing the dimer fusion protein according to claim 1, characterized in that, Includes the following steps: (1) Construct plasmids expressing IL-2 / IL-15 chimera and IL-15RαFc; (2) Culture the cells, add the IL-2 / IL-15 chimeric plasmid DNA and IL-15RαFc plasmid DNA solution to the culture medium, and add the transfection reagent for transfection; (3) Add transfection enhancer, culture, centrifuge, and collect protein supernatant.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the dimer fusion protein of claim 1.

4. The nucleic acid molecule according to claim 3, characterized in that, When the dimer fusion protein is QHJ-02V03, the ORF sequence encoding the IL-2 / IL-15 chimera is shown in SEQ ID NO:12; the ORF sequence encoding IL-15Ra Fc is shown in SEQ ID NO:13; or a sequence having at least 70% sequence identity with the sequences shown in SEQ ID NO:12-13; When the dimer fusion protein is QHJ-02V05, the ORF sequence encoding the IL-2 / IL-15 chimera is shown in SEQ ID NO:14; the ORF sequence encoding IL-15Ra Fc is shown in SEQ ID NO:13; or a sequence having at least 70% sequence identity with the sequences shown in SEQ ID NO:13-14. When the dimer fusion protein is QHJ-02V13, the ORF sequence encoding the IL-2 / IL-15 chimera is shown in SEQ ID NO:15; the ORF sequence encoding IL-15Ra Fc is shown in SEQ ID NO:13; or a sequence having at least 70% sequence identity with the sequences shown in SEQ ID NO:13 and SEQ ID NO:

15. When the dimer fusion protein is QHJ-02V15, the ORF sequence encoding the IL-2 / IL-15 chimera is shown in SEQ ID NO:15; the ORF sequence encoding IL-15Ra Fc is shown in SEQ ID NO:16; or a sequence having at least 70% sequence identity with the sequences shown in SEQ ID NO:15-16. When the dimer fusion protein is QHJ-02M02, the ORF sequence encoding the IL-2 / IL-15 chimera is shown in SEQ ID NO:15; the ORF sequence encoding IL-15Ra Fc is shown in SEQ ID NO:17; or a sequence having at least 70% sequence identity with the sequences shown in SEQ ID NO:15 and SEQ ID NO:

17.

5. An expression carrier, characterized in that, The expression vector carries the nucleic acid molecule as described in any one of claims 3-4.

6. A host cell, characterized in that, The host cell includes: the nucleic acid molecule according to any one of claims 3-4; or the expression vector according to claim 5.

7. The use of the novel dimer fusion protein of claim 1, the nucleic acid molecule of any one of claims 3-4, the expression vector of claim 5, or the host cell of claim 6 in the preparation of a drug for treating cancer.

8. The application according to claim 7, characterized in that, The cancers mentioned include melanoma, lung cancer, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, T-cell lymphoma, B-cell lymphoma, liver cancer, kidney cancer, small cell lung cancer, non-small cell lung cancer, nasopharyngeal carcinoma, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, acute myeloid leukemia, cervical cancer, thyroid cancer, lymphoma, neuroblastoma, brain tumor, myeloma, and glioma.

9. The application according to claim 8, characterized in that, The cancer in question is melanoma.

10. The use of the novel dimer fusion protein of claim 1, or the nucleic acid molecule of any one of claims 3-4, or the expression vector of claim 5, or the host cell of claim 6, in the preparation of a drug for inhibiting lung metastasis of melanoma.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the novel dimer fusion protein of claim 1, or the nucleic acid molecule of any one of claims 3-4, or the expression vector of claim 5, or the host cell of claim 6.

12. The pharmaceutical composition according to claim 11, characterized in that, The pharmaceutical composition may also include a pharmaceutically acceptable carrier.

13. The pharmaceutical composition according to claim 12, characterized in that, The pharmaceutically acceptable carrier is selected from one or more of the following: excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, and flavoring agents.

14. The pharmaceutical composition according to claim 11, characterized in that, The dosage forms of the drug include those administered via the gastrointestinal tract and those administered via non-gastrointestinal tract.

15. The pharmaceutical composition according to claim 14, characterized in that, The drug is administered in a non-gastrointestinal dosage form, which includes: injectable dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

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