Heterodimer fusion protein and application thereof

Abstract

The invention discloses a heterodimer fusion protein and application thereof. The heterodimer fusion protein comprises a first heavy chain and a second heavy chain, wherein the first heavy chain comprises an Fc region and an immunomodulator fused with the Fc region; a light chain and a second heavy chain, which are complexed to form a targeting moiety that exhibits binding specificity for tumor antigens or immune checkpoints; the light chain, the first heavy chain and the second heavy chain are compounded to form the heterodimer fusion protein, and the immunomodulator in the first heavy chain can contain mutations. The heterodimer fusion protein has relatively high affinity to a tumor-associated targeting antigen and an IL-10 receptor, and has good anti-tumor activity.

Description

Heterodimer fusion protein and its application Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a heterodimer fusion protein and applications thereof. Background Art

[0002] IL-10 is primarily secreted by activated T cells and antigen-presenting cells. During antigen recognition, IL-10 receptor (IL-10R) expression is upregulated in CD8+ T cells. IL-10 mediates multiple activities through a specific cell surface receptor complex. The IL-10 receptor consists of two distinct chains, IL-10R1 and IL-10R2, both of which belong to the class II cytokine receptor family (CRF2). During bacterial infection and tissue injury, IL-10 can reduce inflammatory responses, inhibit inflammatory responses induced by T cells (Th17) and macrophages (IL-12 / 23), and reduce tumor-associated inflammatory responses. Within the tumor microenvironment, IL-10 can efficiently activate the proliferation and cytotoxicity of antigen-specific CD8+ T cells.

[0003] The anti-tumor mechanism of IL-10 is as follows: a. It can activate the activity and expansion of CD8+ T cells within tumors; b. IL-10 can increase the activity and expansion of antigen-specific T lymphocytes within tumors; c. IL-10's tumor rejection effect has a memory function. In vivo animal data show that after tumors disappear after IL-10 administration, mice re-inoculated with tumor cells no longer grow in the mice. The main reason is that IL-10 enhances the survival of antigen-specific CD8+ T cells, acting as a tumor vaccine; d. IL-10 reactivates T cells to kill tumor cells by restoring oxidative phosphorylation metabolism in terminally exhausted T cells. Clinical trials have also demonstrated that when used in combination with PDL1 antibodies, it increases the number of PDL1-specific CD8+ positive cells within tumors, resulting in a long-lasting anti-tumor effect. However, there are currently no marketed drugs targeting IL-10. IL-10 can promote the expansion and survival of antigen-specific CD8+ T cells, and antigen-specific CD8+ T cells are positively correlated with immune cell tumor killing. Although multiple studies have demonstrated that immunomodulators can be used to exert anti-tumor effects in animal models and cancer patients, their short half-life and systemic toxicity have greatly limited their use. After IL-10 binds to its receptor, it activates the STAT3 and STAT1 pathways, which are the signal transduction pathways for IL-10's biological functions.

[0004] Patent CN201380041222.1 provides a fusion protein containing unmutated IL-10. However, this fusion protein has low expression levels, low purity, weak IL-10 binding activity and signal activation, and poor binding activity to cancer cells, making it unsuitable for large-scale industrial production. Therefore, it is necessary to find a protein that has good binding activity for both IL-10 and tumor-associated antigens.

[0005] Summary of the Invention

[0006] The present invention aims to provide a heterodimeric fusion protein and its application. The heterodimeric fusion protein has high affinity for tumor antigens or immune checkpoints and IL-10 receptors and has good anti-tumor activity.

[0007] The present invention provides a heterodimeric fusion protein, which comprises: a first heavy chain, wherein the first heavy chain comprises an Fc region and an immunomodulator fused to the Fc region; a light chain and a second heavy chain, wherein the light chain and the second heavy chain are complexed to form a targeting portion that exhibits binding specificity to a tumor antigen or an immune checkpoint; the light chain, the first heavy chain, and the second heavy chain are complexed to form the heterodimeric fusion protein, wherein the immunomodulator in the first heavy chain may contain a mutation.

[0008] In one embodiment, the immunomodulator is a cytokine, a cytokine receptor, a growth factor, a hormone, or an extracellular matrix molecule. In one embodiment, the immunomodulator is selected from IL-1, IL-2, IL-2Rα, IL-2Rβ, IL-3, IL-3Rα, IL-4, IL-4Rα, IL-5, IL-5Rα, IL-6, IL-6Rα, IL-7, IL-7Rα, IL-8, IL-9, IL-9Rα, IL-10, IL-10R1, IL-10R2, IL-11, IL-11Rα, IL-12, IL-12R One or more of IL-13Rα, IL-12Rβ2, IL-12Rβ1, IL-13, IL-13Rα, IL-13Rα2, IL-14, IL-15, IL-15Rαsushi, IL-16, IL-17, IL-18, IL-19, IL-20, IL-20R1, IL-20R2, IL-21, IL-21Rα, IL-22, IL-23, IL-23R, IL-27R, and IL-31R. In one embodiment, the immunomodulator is IL-10.

[0009] In one embodiment, the amino acid sequence of IL-10 is as shown in SEQ ID NO: 1 or 2, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 or 2.

[0010] In one embodiment, the amino acid sequence of IL-10 is as shown in SEQ ID NO: 1, which may contain one or more of the following mutation sites: N18Y, R104W, N92Q, T100D.

[0011] In one embodiment, the tumor antigen or immune checkpoint is B7H3, B7H4, B7H5, BTLA, CD27, CD28, CD153, CD40, CD40L, CD70, CD80, CD86, CD96, CD112, CD134, CD137, CD137L, CD152 / CTLA-4, CD155, CD223, CD226, CD252 / OX40L, CD258, CD273 / PD-L2, CD274 / PD-L1, CD278, CD279, CD357, DR3, Galectin-9, GITRL, HVEM, ICOSL / B7RP1 / B7H2, IDO, TIGIT, TIM-3, T L1A, MART-1 / MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, CEA, p53, Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen EBVA, human papillomavirus antigen E6 or E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, erbB, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\Cyclophilin C-related protein, TAAL6, TAG72, TLP, MUC16, IL13Rα2, FRα, VEGFR2, Lewis Y, FAP, EphA2, CEACAM5, CEACAM6, EGFR, CA6, CA9, GPNMB, EGP1, FOLR1, endothelial receptor, STEAP1, SLC44A4, integrin-4, One or more of AGS-16, guanidinyl cyclase C, MUC-1, CFC1B, integrin α3 chain, TPS, CD19, CD20, CD22, CD30, CD72, CD180, CD171, CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, CLL-1 / CLEC12A, ROR1, glypican 3, mesothelin, CD33 / IL3Ra, c-Met, PSCA, PSMA, glycolipid F77, EGFRvIII, BCMA, GD-2, MY-ESO-1, or MAGE A3.

[0012] In one embodiment, the light chain comprises a complementarity determining region (CDR) comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of the corresponding CDR of the light chain of an antibody that specifically binds to a tumor antigen or an immune checkpoint.

[0013] In one embodiment, the light chain of the antibody that specifically binds to a tumor antigen or an immune checkpoint contains LCDR1 with an amino acid sequence as shown in SEQ ID NO:6, LCDR2 with an amino acid sequence as shown in SEQ ID NO:7, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:8.

[0014] In one embodiment, the light chain comprises a variable region comprising an amino acid sequence that is at least 80% identical to an amino acid sequence comprised in a light chain variable region of an antibody specific for a tumor antigen or an immune checkpoint.

[0015] In one embodiment, the amino acid sequence of the variable region of the light chain is as shown in SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 10.

[0016] In one embodiment, the amino acid sequence of the light chain is as shown in SEQ ID NO: 13, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 13.

[0017] In one embodiment, the second heavy chain comprises a complementarity determining region (CDR), which comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the corresponding CDR of the second heavy chain of an antibody that specifically binds to a tumor antigen or an immune checkpoint.

[0018] In one embodiment, the second heavy chain of the antibody that specifically binds to a tumor antigen or an immune checkpoint comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO: 3, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 5.

[0019] In one embodiment, the second heavy chain comprises a variable region comprising an amino acid sequence that is at least 80% identical to an amino acid sequence comprised in a light chain variable region of an antibody specific for a tumor antigen or an immune checkpoint.

[0020] In one embodiment, the amino acid sequence of the variable region of the second heavy chain is as shown in SEQ ID NO: 9, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 9.

[0021] In one embodiment, the amino acid sequence of the second heavy chain is as shown in SEQ ID NO: 12, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 12.

[0022] In one embodiment, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO: 15, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 15.

[0023] In one embodiment, the immunomodulator is linked to the Fc region of the antibody that specifically binds to a tumor antigen or an immune checkpoint.

[0024] In one embodiment, the first heavy chain comprises a constant region of an immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

[0025] In one embodiment, the first heavy chain contains one or more Fc regions of the same or different types, and the Fc region is fused to the immunomodulator via a polypeptide linker. In one embodiment, the immunomodulator is connected to the N-terminus of the Fc region via a polypeptide linker. In one embodiment, the polypeptide linker is 5-30 amino acids. In one embodiment, the polypeptide linker is (GGGGS)n, where n=1-6.

[0026] In one embodiment, the first heavy chain comprises one or more immunomodulatory agents of the same or different types, fused to each other and to the Fc region.

[0027] The present invention also provides a method for preparing the aforementioned heterodimeric fusion protein, comprising transferring three recombinant plasmids containing the aforementioned light chain, first heavy chain, and second heavy chain into the same host cell for recombinant expression. In one embodiment, the host cell is a mammalian cell, a bacterium, a fungus, or an insect cell. In one embodiment, the mammalian cell is a CHO cell, an SP20 cell, an NSO cell, a COS cell, a BHK cell, a HEK293 cell, or a PerC6 cell. In one embodiment, the mammalian cell is a CHO cell.

[0028] The present invention also provides a nucleic acid encoding the heterodimeric fusion protein, a vector or plasmid containing the nucleic acid, and a cell expressing the vector or plasmid.

[0029] The present invention also provides a pharmaceutical composition comprising the aforementioned heterodimeric fusion protein and at least one pharmaceutically acceptable excipient, diluent, or carrier. In one embodiment, the pharmaceutical composition can be used alone or in combination with other therapeutic agents to enhance efficacy or reduce potential side effects.

[0030] The present invention also provides the use of the heterodimeric fusion protein in the preparation of a medicament for treating a tumor disease. In one embodiment, the tumor disease comprises one or more of colorectal cancer, colorectal cancer, pancreatic cancer, lung cancer, esophageal cancer, prostate cancer, desmoplastic small round cell tumor, ovarian cancer, gastric cancer, pancreatic cancer, liver cancer, kidney cancer, breast cancer, non-small cell lung cancer, melanoma, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, Ewing's sarcoma, Wilms' tumor, neuroblastoma, ganglioneuroma, medulloblastoma, high-grade glioma, diffuse intrinsic pontine glioma, and multi-layered rosette embryonal tumor.

[0031] The present invention also provides the use of the above-mentioned heterodimeric fusion protein in preparing a reagent or a kit for detecting tumor antigens or immune checkpoints and IL-10 receptor molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 : Schematic diagram of the structure of the heterodimeric fusion protein of the present invention.

[0033] Figure 2: ELISA detection of the binding activity of fusion proteins 1 and 2 to HER2 protein.

[0034] Figure 3: ELISA detection of the binding activity of fusion protein 2 to IL-10 receptor protein.

[0035] FIG4 shows the binding activity of the heterodimeric fusion protein of the present invention to BT474 cells. FIG5 shows the verification of IL-10 terminal luciferase expression activation in positive and negative controls.

[0036] Figure 6: Verification of IL-10 end luciferase expression activation of fusion protein 1.

[0037] Figure 7: Verification of IL-10 end luciferase expression activation of fusion protein 2. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with Examples, which are used to describe some specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the application belongs. Although methods and materials similar or equivalent to those described herein can be used for the practice or test of the application, suitable methods and materials are described below. In the event of a conflict, the patent specification shall prevail.

[0039] The term "heterodimer" generally refers to a molecule (e.g., a protein molecule) composed of two different members. The two members of a heterodimer may differ in structure, function, activity, and / or composition. For example, the two different members may comprise polypeptides that differ in the order, number, or type of amino acid residues that form these polypeptides. Each of the two different members of a heterodimer may independently comprise one, two, or more units, polypeptide chains, or portions.

[0040] The term "targeting moiety" generally refers to a molecule, complex or aggregate that specifically, selectively or preferentially binds to a target molecule, cell, particle, tissue or aggregate. For example, the targeting moiety can be an antibody, an antigen-binding antibody fragment, a bispecific antibody or other antibody-based molecule or compound. Other examples of targeting moieties can include, but are not limited to, aptamers, high-affinity polymers, receptor-binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides or proteins, etc.

[0041] The term "antigen binding site" or "binding portion" generally refers to a part of an antibody that participates in antigen binding. The antigen binding site can be formed by amino acid residues in the N-terminal variable ("V") region of the heavy ("H") chain and / or light ("L") chain. The three highly divergent segments within the V region of the heavy and light chains are referred to as "hypervariable regions," which are inserted between more conservative flanking segments called "framework regions" or "FRs." In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen binding "surface." This surface can mediate the recognition and binding of the target antigen.

[0042] There are a variety of methods / systems in the art to define and describe CDRs, and these systems and / or definitions have been developed and refined over the years, including Kabat, Chothia, IMGT, AbM, and Contact. Kabat is the most commonly used and defines CDRs based on sequence variability; Chothia defines CDRs based on sequence variability based on the position of the structural loop region; the IMGT system defines CDRs based on sequence variability and position within the variable domain structure; AbM is based on the AbM antibody modeling software from Oxford Molecular and is a compromise between Kabat and Chothia; Contact defines CDRs based on the analysis of complex crystal structures and is similar to Chothia in many aspects. The numbering of amino acid positions (e.g., amino acid residues in the Fc region) and target regions (e.g., CDRs) in the present invention uses the Kabat system.

[0043] The term "tumor antigen" generally refers to an antigenic substance produced in or by a tumor cell, which may have the ability to trigger an immune response in a host. For example, a tumor antigen may be a protein, polypeptide, peptide, or fragment thereof that constitutes a part of a tumor cell and is capable of inducing tumor-specific cytotoxic T lymphocytes. In some embodiments, the term "tumor antigen" may also refer to a biological molecule (e.g., protein, carbohydrate, glycoprotein, etc.) that is uniquely or preferentially or differentially expressed on cancer cells and / or is found to be associated with cancer cells, thereby providing a preferential or specific target for cancer. For example, preferential expression may be preferential expression compared to any other cell in an organism, or preferential expression within a specific region of an organism (e.g., within a specific organ or tissue).

[0044] The term "immune checkpoint" generally refers to inhibitory and activating molecules in the immune system that regulate the body's anti-tumor immune system by modulating T cell activity. For example, inhibitory molecules include PDL1, B7H3, and CTLA4, while activating molecules include OX40, 4-1BB, and CD40.

[0045] The term "immunomodulator" generally refers to a substance that affects the function of the immune system. An immunomodulator can enhance or reduce an immune response. For example, an immunomodulator can be an active agent of immunotherapy, including but not limited to, for example, cytokines, granulocyte colony stimulating factor (G-CSF), interferon, imiquimod, cell membrane fragments from bacteria, chemokines, interleukins, cytosine phosphate-guanosine (CpG) oligodeoxynucleotides and recombinant, synthetic and / or natural preparations of dextran. In some embodiments, the immunomodulator is a cytokine.

[0046] The term "polypeptide linker" generally refers to a synthetic amino acid sequence that connects or couples two polypeptide sequences (e.g., connects two polypeptide domains). A polypeptide linker can connect two amino acid sequences via a peptide bond. In some embodiments, the polypeptide linker of the present application connects an immunomodulator to an Fc region.

[0047] The term "antibody" generally refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes or immunoglobulin gene fragments. Immunoglobulin genes can include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. As used herein, light chains can be classified as kappa or lambda. Heavy chains can be classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively. The antibodies used in this application can have structural units comprising tetramers. Each tetramer can be composed of two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each member can define a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The terms light chain variable region (VL) and heavy chain variable region (VH) generally refer to these regions of the light and heavy chains, respectively. Antibodies can exist as complete immunoglobulins or as many well-characterized fragments produced by digestion with various peptidases or de novo expression. The term "antibody" can also include antibody fragments produced by modifying whole antibodies or using recombinant DNA methods to synthesize de novo, including but not limited to Fab'2, IgG, IgM, IgA, IgE, scFv, dAb, nano antibodies, single antibodies and double-chain antibodies. In some embodiments, antibodies include but are not limited to Fab'2, IgG, IgM, IgA, IgE and single-chain antibodies, such as single-chain Fv (scFv) antibodies, in which the variable heavy chain and the variable light chain are linked together (directly or through a peptide linker) to form a continuous polypeptide. In some embodiments, the antibodies and fragments in this application are bispecific. In some embodiments, bispecific antibodies or fragments thereof have binding specificity to at least two different epitopes (for example, at least one of the at least two different epitopes is a tumor-associated antigen). In some embodiments, antibodies and fragments can also be heterologous antibodies, for example, they can be or can comprise two or more antibodies or antibody binding fragments (such as Fab) linked together, wherein each antibody or fragment has different specificities.

[0048] The term "identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity. Comparisons for purposes of determining percent amino acid sequence identity can be performed in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST software or the FASTA program package.

[0049] The term "at least 80% identity" means that the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence is greater than 80%, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0050] The term "host cell" generally includes individual cells, cell lines or cell cultures that may be or have been recipients of a subject's plasmid or vector, comprising polynucleotides disclosed herein, or expressing heterodimeric proteins of the present application. Host cells may include offspring of a single host cell. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical (morphologically or in genomic total DNA complement) to the original parental cell. Host cells may include cells transfected in vitro with a vector disclosed herein. Host cells may be bacterial cells (e.g., Escherichia coli (E. coli)), yeast cells or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells or myeloma cells.

[0051] The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into a host cell and / or between host cells. The term can include vectors primarily used to insert DNA or RNA into cells, vectors primarily used for replication of DNA or RNA, and expression vectors used for transcription and / or translation of DNA or RNA. Vectors that provide more than one of the above functions are also included. An "expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell.

[0052] The term "treatment" refers to an approach for obtaining a beneficial or desired result, including but not limited to a therapeutic benefit and / or a preventive benefit. A therapeutic benefit generally refers to eradication or lessening of the severity of the underlying condition being treated. In addition, a therapeutic benefit is achieved by eradicating, lessening the severity, or reducing the incidence of one or more physiological symptoms associated with the underlying condition such that an improvement is observed in the subject (although the subject may still be afflicted with the underlying condition). For preventive benefit, a composition may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.

[0053] The term "in vivo" generally refers to events that occur within the body of a subject.

[0054] The term "in vitro" generally refers to an event that occurs outside the body of a subject. For example, an in vitro assay includes any assay performed outside a subject. In vitro assays include cell-based assays in which dead or live cells are used. In vitro assays also include cell-free assays in which intact cells are not used.

[0055] The term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, goat, rabbit, mouse, rat, or monkey.

[0056] The amino acid sequences of the heterodimeric fusion proteins herein (hereinafter referred to as "fusion proteins"), IL-10 and its variants, and the control antibodies mentioned in the Examples are shown in Table 1. The structures of fusion proteins 1 and 2 are shown in Figure 1 , with the only difference being that IL-10 in fusion protein 1 is native, while IL-10 in fusion protein 2 is mutated.

[0057] Table 1. Amino acid sequences of fusion proteins, IL10, etc. of the present invention

[0058] Example 1 Gene synthesis and construction of expression vector

[0059] The PcDNA3.1 vector is used as a dedicated vector for expressing the light and heavy chains of the fusion protein. The PcDNA3.1 vector contains the CMV Promoter promoter used by the heavy chain, the eukaryotic screening marker G418 tag, and the prokaryotic screening tag Ampicilline. Gene synthesis obtains the nucleotide sequences of the first heavy chain, second heavy chain, and light chain encoding genes (i.e., target genes) expressed by the fusion protein. The vector and target fragments are double-digested with HindIII and XhoI, and then enzymatically linked with DNA ligase after recovery. The recombinant plasmid containing the genes encoding the first heavy chain, second heavy chain, and light chain of the fusion protein is transformed into Escherichia coli competent cells DH5α, and the positive clones are selected and subjected to plasmid extraction and enzyme digestion verification to obtain a recombinant plasmid containing the genes encoding the first heavy chain, second heavy chain, and light chain of the fusion protein.

[0060] Example 2 Plasmid Extraction

[0061] According to the method described in the Molecular Cloning Experimental Guide (2002, Science Press), the recombinant plasmids containing the above-mentioned target genes were transformed into competent Escherichia coli DH5α cells. The transformed bacteria were spread on LB plates containing 100 μg / mL ampicillin and cultured. The selected plasmid clones were cultured in liquid LB medium and shaken at 260 rpm for 14 h. The plasmids were extracted using an endotoxin-free plasmid extraction kit, dissolved in sterile water, and the concentration was measured using a nucleic acid protein quantifier.

[0062] Example 3 Plasmid transfection, transient expression and fusion protein purification

[0063] Culture ExpiCHO at 37°C, 8% CO2, and 100 rpm to a cell density of 6 × 10 6 The constructed vector plasmids were transfected into the above cells using liposomes at a mass concentration of 1:1:1. The transfection plasmid concentration was 1 mg / mL. The liposome concentration was based on the ExpiCHO TM As determined by the Expression System kit, culture was performed at 32°C, 5% CO2, and 100 rpm for 7-10 days. Feeds were added 18-22 hours after transfection and again on day 5. The culture product was centrifuged at 4000 g, filtered through a 0.22 μm filter, and the supernatant was collected. The resulting antibody protein was purified using a Protein A column and an ion column, and the eluate was collected.

[0064] The specific operation steps of ProteinA and ion column purification are as follows: after high-speed centrifugation of the cell culture fluid, the supernatant is taken and affinity chromatography is performed using GE's ProteinA chromatography column. The equilibration buffer used for chromatography is 1×PBS (pH 7.4). After the cell supernatant is loaded and bound, it is washed with PBS until the ultraviolet light returns to the baseline, and then the target protein is eluted with 0.1M glycine (pH 3.0) in the elution buffer, and the pH is adjusted to neutral for storage using Tris. The pH of the product obtained by affinity chromatography is adjusted to 1-2 pH units lower or higher than the isoelectric point pI, and appropriately diluted to control the sample conductivity below 5ms / cm. Using appropriate corresponding pH buffers such as phosphate buffer, acetate buffer and other conditions, conventional ion exchange chromatography methods in the art such as anion exchange or cation exchange are used to perform NaCl gradient elution under corresponding pH conditions, and the collection tube where the target protein is located is selected according to SDS-PAGE and stored together. Then, the eluate obtained after purification is ultrafiltered and exchanged into the buffer.

[0065] Example 4 ELISA detection of affinity of fusion protein for HER2

[0066] huHER2-his (purchased from ACRO, CAT: HE2-H5225) was diluted to 0.5 μg / mL in PBS buffer (pH 7.4). 100 μL was added to each well of a 96-well ELISA plate and coated overnight at 4°C. The plate was blocked with 1% BSA blocking buffer for 1 hour. After washing the plate three times with PBST, the purified fusion protein was diluted to 100 nM in 0.5% BSA sample diluent. This was used as the starting concentration for a three-fold serial dilution, resulting in 11 steps. A negative control (NEO-201 antibody, msIgG1 subtype, see CN111670199A, heavy chain amino acid sequence: SEQ ID NO: 16, light chain amino acid sequence: SEQ ID NO: 17) and a positive control antibody (INN trastuzumab) were added to each well at 100 μL and incubated at 37°C for 1 hour. Wash the plate three times with PBST and add 100 μL of HRP-conjugated goat anti-human IgG Fc (Jackson Cat: 109-035-098) to each well at a 1:10,000 dilution in sample diluent. Incubate at room temperature for 1 hour. Wash the plate four times with PBST and add 100 μL of TMB substrate to each well. Incubate at room temperature in the dark for 10 minutes. Terminate the color development reaction by adding 100 μL of 1 M HCl to each well. Measure the absorbance of each well in the 96-well plate on a multi-functional microplate reader at a wavelength of 450 nm and a reference wavelength of 570 nm. The absorbance (OD) of each well = OD450 nm - OD570 nm. The logarithm of the fusion protein concentration was used as the abscissa, and the measured absorbance value of each well was used as the ordinate. Sigmoidal dose-response (Variable Slope) method (Graph Pad Prism software, Graph Pad Software, San Diego, California) was used for nonlinear regression to obtain the binding curve of the fusion protein and HER2 protein.

[0067] The ELISA results of the fusion protein are shown in FIG2 , and the fusion protein 2 can bind to HER2 at multiple concentration ranges.

[0068] Example 5 ELISA detection of affinity of fusion protein for IL-10 receptor

[0069] The IL-10 receptor human IL10RA-his (purchased from Sino Biological, Beijing, CAT: 10419-H08H) was diluted to 0.8 μg / mL using PBS buffer at pH 7.4. 100 μL was added to each well of a 96-well ELISA plate and coated overnight at 4°C. Blocking was performed with 1% BSA blocking buffer for 1 hour. After washing the plate three times with PBST, the purified antibody was diluted to 100 nM using 0.5% BSA sample diluent. This was used as the starting concentration for a three-fold gradient dilution, totaling 11 steps. A negative control (INN trastuzumab) and a positive control (the fusion protein obtained in Example 4 of Patent 202110141918.8) were set up, with 100 μL per well added and incubated at 37°C for 1 hour. Wash the plate three times with PBST and add 100 μL of HRP-conjugated goat anti-human IgG Fc (Jackson Cat: 109-035-098) to each well at a 1:10,000 dilution in sample diluent. Incubate at room temperature for 1 hour. Wash the plate four times with PBST and add 100 μL of TMB substrate to each well. Incubate at room temperature in the dark for 10 minutes. Terminate the color reaction by adding 100 μL of 1 M HCl to each well.

[0070] The absorbance of each well in a 96-well plate was measured on a multifunctional microplate reader at a wavelength of 450 nm and a reference wavelength of 570 nm. The absorbance per well (OD) = OD450 nm - OD570 nm. The logarithm of the antibody concentration was plotted as the abscissa, and the measured absorbance per well as the ordinate. Sigmoidal dose-response (Variable Slope) method (Graph Pad Prism software, Graph Pad Software, San Diego, California) was used for nonlinear regression to generate the binding curve between the target fusion protein and the IL-10 receptor IL-10RA protein.

[0071] The ELISA results of the fusion protein are shown in FIG3 . Fusion protein 2 can bind to the IL-10 receptor in multiple concentration ranges, which is better than the positive control.

[0072] Example 6 Binding activity of the IL-10 bioactive HER2 end of the fusion protein to BT474 cells

[0073] Take logarithmic growth of BT474 cells (from Shanghai Chinese Academy of Sciences) with normal morphology, transfer to a centrifuge tube and centrifuge at 1000 rpm for 5 min. 6Resuspend the cells at a density of 100 μL / mL in FACS buffer, and aliquot 100 μL of cells into separate tubes. Dilute the purified fusion protein to 200 nM in FACS buffer. Using this as the starting concentration, perform a 5-fold serial dilution, totaling eight steps. A negative control (i.e., the NEO-201 antibody msIgG1 subtype, see CN 111670199A, heavy chain amino acid sequence: SEQ ID NO: 16, light chain amino acid sequence: SEQ ID NO: 17) and a positive control (INN trastuzumab) were also added, along with 100 μL of the fusion protein dilution. The cells were incubated at 4°C for 60 minutes and then washed twice with excess FACS buffer. The cells were resuspended in 100 μL of FACS buffer, and a secondary goat anti-human PE antibody (Biolgend, Cat: 398004) was added to the sample, incubated for 30 minutes, and washed twice with excess FACS buffer. The cells were fixed in fixation buffer and subsequently analyzed by flow cytometry. The binding activity of the fusion protein to BT474 cells was detected by FACS.

[0074] The FACS detection results of the binding activity of fusion proteins 1 and 2 to BT474 cells are shown in FIG4 . Both fusion proteins 1 and 2 can specifically bind to BT474 at multiple concentration ranges.

[0075] Example 7 Verification of IL-10 terminal luciferase expression activation

[0076] IL-10 binds to the IL-10R, mediating control over the extent and duration of inflammation, which is crucial for maintaining homeostasis in the body's inflammatory response. This process relies on the regulation of signal transducer and activator of transcription 3 (STAT3). This experiment used HEK293 tool cells, which stably express the IL-10R and STAT3 signaling pathway reporter gene systems. Stimulation with IL-10 protein activated an increase in luciferase expression in the cells. Therefore, this cell line was used for studying IL-10-STAT3 activity.

[0077] IL-10-Reporter-HEK-293 cell lines in the logarithmic growth phase (purchased from Jiman Bio) were used, and 2.5×10 4Cells were plated in a 96-well cell culture plate (WHB, Cat: WHB-96-01) and cultured overnight in a 37°C, 5% CO2 incubator. The positive control (fusion protein obtained in Example 4 of Patent 202110141918.8), IL-10 protein (Novoprotein, Cat: CX04), negative control (cetuximab), human IgG1, fusion protein 1, and fusion protein 2 were each diluted fourfold starting at 300 μM in complete DMEM medium, for a total of nine concentrations. 100 μL / well of each dilution was added to the 96-well cell culture plate after the supernatant was aspirated. After mixing, the plates were incubated in a 37°C, 5% CO2 incubator for 6 hours. Then, 100 μL / well of Bio-Lite Luciferase Assay Substrat (Vazyme, Cat: DD1201-02) colorimetric solution was added and incubated at room temperature for 10 minutes. Then, the fluorescence value was detected using an enzyme-labeled instrument.

[0078] The fusion protein concentration was used as the horizontal axis and the relative luminescence value (RLU) was used as the vertical axis. The nonlinear regression analysis method in GraphPad Prism software (Graphpad Prism 5 Demo, San Diego, California) was used to fit the curve graph using the Log (agonist) vs Response--Variable Slope method to obtain the fusion protein fluorescence dose-effect curve.

[0079] As shown in Figure 5, IL-10 and the positive control activated luciferase expression in HEK293 cells in a dose-dependent manner, while the negative control cetuximab and human IgG1 had no such effect, indicating that this reporter gene method can reflect the signal activation activity of IL-10. Calculate the positive control EC 50 / fusion protein EC 50 If the relative activity value is greater than 100%, it means that the activity of the fusion protein is better than that of the positive control. Otherwise, the activity of the fusion protein is lower than that of the positive control. 50 The EC values ​​of the positive control and fusion protein 2 were 2.259 nM and 5.839 nM, respectively, and the relative activity values ​​of the two were 39%. 50 The relative activity values ​​of the two proteins were 2.117 nM and 0.648 nM, respectively, and were 327%. It can be seen that the activation activity of fusion protein 2 is better than that of fusion protein 1.

[0080] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A heterodimeric fusion protein, characterized in that The heterodimeric fusion protein comprises: a first heavy chain, wherein the first heavy chain comprises an Fc region and an immunomodulator fused to the Fc region; a light chain and a second heavy chain, wherein the light chain and the second heavy chain are complexed to form a targeting portion that exhibits binding specificity to a tumor antigen or an immune checkpoint; the light chain, the first heavy chain, and the second heavy chain are complexed to form the heterodimeric fusion protein, wherein the immunomodulator in the first heavy chain may contain a mutation.

2. The heterodimeric fusion protein according to claim 1, characterized in that The immunomodulator is a cytokine, a cytokine receptor, a growth factor, a hormone or an extracellular matrix molecule; preferably, the immunomodulator is selected from IL-1, IL-2, IL-2 Rα, IL-2 Rβ, IL-3, IL-3 Rα, IL-4, IL-4 Rα, IL-5, IL-5 Rα, IL-6, IL-6 Rα, IL-7, IL-7 Rα, IL-8, IL-9, IL-9 Rα, IL-10, IL-10R1, IL-10R2, IL-11, IL-11 Rα, IL-12, IL-12 Rα, IL-12 Rβ2, IL-12 Rβ1, IL-13, IL-13 Rα, IL-13 Rα2, IL-14, IL-15, IL-15Rαsushi, IL-16, IL-17, IL-18, IL-19, IL-20, IL-20R1, IL-20R2, IL-21, IL-21 Rα, IL-22, IL-23, IL-23R, IL-27 R, IL-31 R; more preferably, the immunomodulator is IL-10.

3. The heterodimeric fusion protein according to claim 1 or 2, characterized in that The immunomodulator comprises at least 1-4 mutations.

4. The heterodimeric fusion protein according to claim 3, characterized in that The mutation site includes one or more of N18Y, R104W, N92Q, and T100D.

5. The heterodimeric fusion protein according to any one of claims 1 to 4, characterized in that The first heavy chain contains one or more immunomodulators, which are fused to each other and to the Fc region; preferably, the immunomodulator is IL-10; more preferably, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO: 15, or an amino acid sequence that has at least 80% identity with SEQ ID NO:

15.

6. The heterodimeric fusion protein according to any one of claims 1 to 5, characterized in that The tumor antigens or immune checkpoints are B7H3, B7H4, B7H5, BTLA, CD27, CD28, CD153, CD40, CD40L, CD70, CD80, CD86, CD96, CD112, CD134, CD137, CD137L, CD152 / CTLA-4, CD155, CD223, CD226, CD252 / OX40L, CD258, CD273 / PD-L2, CD274 / PD-L1, CD278, CD279, CD357, DR3, Galectin-9, GITRL, HVEM, ICOSL / B7RP1 / B7H2, IDO, TIGIT, TIM-3, and TL1A. , MART-1 / MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, CEA, p53, Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen EBVA, human papillomavirus antigen E6 or E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, erbB, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, MUC16, IL13Rα2, FRα, VEGFR2, Lewis Y, FAP, EphA2, CEACAM5, CEACAM6, EGFR, CA6, CA9, GPNMB, EGP1, FOLR1, endothelial receptor, STEAP1, SLC44A4, integrin-4, AGS-16, guanidinyl cyclase C, MUC-1, CFC1B, integrin α3 chain, TPS, CD19, CD20, CD22, CD30, CD72, CD180, CD171, One or more of CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, CLL-1 / CLEC12A, ROR1, Glypican 3, Mesothelin, CD33 / IL3Ra, c-Met, PSCA, PSMA, glycolipid F77, EGFRvIII, BCMA, GD-2, MY-ESO-1, or MAGE A3.

7. The heterodimeric fusion protein according to claim 6, characterized in that Both the light chain and the second heavy chain contain complementary determining regions, and the complementary determining regions contain an amino acid sequence that is at least 80% identical to the amino acid sequence of the corresponding CDR of the light chain or heavy chain of an antibody that specifically binds to a tumor antigen or an immune checkpoint; preferably, the light chain of the antibody that specifically binds to a tumor antigen or an immune checkpoint contains LCDR1 with an amino acid sequence as shown in SEQ ID NO: 6, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 7, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 8; preferably, the second heavy chain of the antibody that specifically binds to a tumor antigen or an immune checkpoint contains HCDR1 with an amino acid sequence as shown in SEQ ID NO: 3, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:

5.

8. A nucleic acid encoding the heterodimeric fusion protein according to any one of claims 1 to 7, a vector or plasmid containing the nucleic acid, or a cell expressing the vector or plasmid.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the heterodimeric fusion protein according to any one of claims 1 to 7 and at least one pharmaceutically acceptable excipient, diluent or carrier.

10. Use of the heterodimeric fusion protein according to any one of claims 1 to 7, characterized in that: The applications include: (a) preparing drugs for treating tumor diseases, wherein the tumor diseases include one or more of colorectal cancer, pancreatic cancer, lung cancer, esophageal cancer, prostate cancer, desmoplastic small round cell tumor, ovarian cancer, gastric cancer, pancreatic cancer, liver cancer, kidney cancer, breast cancer, non-small cell lung cancer, melanoma, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, Ewing's sarcoma, Wilms' tumor, neuroblastoma, ganglioneuroma, medulloblastoma, high-grade glioma, diffuse intrinsic pontine glioma, and multi-layered rosette embryonal tumor; or (b) preparing reagents or kits for detecting tumor antigens or immune checkpoints and IL-10 receptor molecules.

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