WO2024008039A1 - 一种异源二聚体融合蛋白及其应用 - Google Patents

一种异源二聚体融合蛋白及其应用 Download PDF

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WO2024008039A1
WO2024008039A1 PCT/CN2023/105535 CN2023105535W WO2024008039A1 WO 2024008039 A1 WO2024008039 A1 WO 2024008039A1 CN 2023105535 W CN2023105535 W CN 2023105535W WO 2024008039 A1 WO2024008039 A1 WO 2024008039A1
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fusion protein
heavy chain
amino acid
acid sequence
cancer
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French (fr)
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周冲
殷刘松
姜晓玲
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盛禾(中国)生物制药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the invention belongs to the technical field of biomedicine, and specifically relates to a heterodimer fusion protein and its application.
  • IL-10 is mainly secreted by activated T cells and antigen-presenting cells. During the process of antigen recognition, the expression of IL-10 receptor (IL-10R) in CD8+ T cells is up-regulated. IL-10 mediates multiple activities through a specific cell surface receptor complex.
  • the IL-10 receptor contains two different chains, IL-10R1 and IL-10R2, both of which belong to the class II cytokine receptor family. (CRF2).
  • IL10 can reduce inflammatory responses, inhibit inflammatory responses caused by T cells (Th17) and macrophages (IL-12/23), and reduce tumor-related inflammatory responses. In the tumor microenvironment, IL-10 can efficiently activate the proliferation and toxicity of antigen-specific CD8+ T cells.
  • the anti-tumor mechanism of IL-10 is: a. It can activate the activity and expansion of CD8+ T cells inside the tumor; b. IL10 can increase the activity and expansion of antigen-specific T lymphocytes inside the tumor; c. IL-10 The rejection of tumors has a memory function. In vivo animal test data shows that after the tumor disappears after the IL-10 drug is given to the mice, the tumor cells are again inoculated into the mice. The tumor cells do not grow in the mice. The main reason is: IL-10 can Enhance the survival rate of antigen-specific CD8+ T cells and act as a tumor vaccine; d. IL10 reactivates T cells by restoring the oxidative phosphorylation metabolism of terminally exhausted T cells to kill tumor cells.
  • Patent CN201380041222.1 provides a fusion protein containing unmutated IL-10.
  • the expression level of this fusion protein is low, the purity is not high, the binding activity and signal activation ability of the IL-10 end are weak, and the binding activity to cancer cells is poor, so it cannot be used for large-scale industrial production. Therefore, it is necessary to find proteins that have good binding activity to both IL-10 and tumor-associated antigens.
  • the object of the present invention is to provide a heterodimer fusion protein and its application.
  • This heterodimeric fusion protein has high affinity for tumor antigens or immune checkpoints and IL-10 receptors, and has good anti-tumor activity.
  • the present invention provides a heterodimer fusion protein, which includes: a first heavy chain that includes an Fc region and an immunomodulator fused to the Fc region; a light chain and a second heavy chain, the light chain and the second heavy chain complexed to form a targeting moiety exhibiting binding specificity for a tumor antigen or immune checkpoint; the light chain, the first heavy chain, the second heavy chain Complexed to form the heterodimeric fusion protein, wherein the immunomodulator in the first heavy chain may contain a mutation.
  • the immunomodulatory agent is a cytokine, cytokine receptor, growth factor, hormone or extracellular matrix molecule.
  • the immunomodulatory agent is selected from the group consisting of IL-1, IL-2, IL-2R ⁇ , IL-2R ⁇ , IL-3, IL-3R ⁇ , IL-4, IL-4R ⁇ , IL-5, IL -5R ⁇ , IL-6, IL-6R ⁇ , IL-7, IL-7R ⁇ , IL-8, IL-9, IL-9R ⁇ , IL-10, IL-10R1, IL-10R2, IL-11, IL-11R ⁇ , IL-12, IL-12R ⁇ , IL-12R ⁇ 2, IL-12R ⁇ 1, IL-13, IL-13R ⁇ , IL-13R ⁇ 2, IL-14, IL-15, IL-15R ⁇ sushi, IL-16, IL-17, IL -18, one of IL-19, IL-20, IL-20R1, IL-20R2, IL-21, IL
  • the amino acid sequence of IL-10 is as shown in SEQ ID NO: 1 or 2, or is an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 or 2.
  • amino acid sequence of IL-10 is shown in SEQ ID NO: 1, which may include one or more of the following mutation sites: N18Y, R104W, N92Q, T100D.
  • 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, 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
  • the light chain comprises a complementarity determining region (CDR) comprising at least 80% identity to the amino acid sequence of the corresponding CDR of the light chain of an antibody that specifically binds a tumor antigen or immune checkpoint amino acid sequence.
  • CDR complementarity determining region
  • the light chain of the antibody that specifically binds to a tumor antigen or 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 LCDR2 with an amino acid sequence as shown in SEQ ID NO:7.
  • the light chain comprises a variable region comprising at least 80% identity to an amino acid sequence comprised in a light chain variable region of an antibody specific for a tumor antigen or immune checkpoint amino acid sequence.
  • the amino acid sequence of the variable region of the light chain is as shown in SEQ ID NO: 10, or is an amino acid sequence that is at least 80% identical to SEQ ID NO: 10.
  • the amino acid sequence of the light chain is as shown in SEQ ID NO: 13, or is an amino acid sequence that is at least 80% identical to SEQ ID NO: 13.
  • the second heavy chain comprises a complementarity determining region (CDR) comprising an amino acid sequence corresponding to the CDR of the second heavy chain of an antibody that specifically binds a tumor antigen or an immune checkpoint having at least Amino acid sequence with 80% identity.
  • CDR complementarity determining region
  • the second heavy chain of the antibody that specifically binds to a tumor antigen or immune checkpoint contains HCDR1 with an amino acid sequence as shown in SEQ ID NO:3, and HCDR2 with an amino acid sequence as shown in SEQ ID NO:4 , the amino acid sequence is HCDR3 shown in SEQ ID NO:5.
  • the second heavy chain comprises a variable region comprising at least 80% identical amino acid sequence to that contained in the light chain variable region of an antibody specific for a tumor antigen or immune checkpoint. Identity of the amino acid sequence.
  • variable region amino acid sequence of the second heavy chain is as shown in SEQ ID NO:9, or is an amino acid sequence having at least 80% identity with SEQ ID NO:9.
  • the amino acid sequence of the second heavy chain is as shown in SEQ ID NO: 12, or is an amino acid sequence that is at least 80% identical to SEQ ID NO: 12.
  • the amino acid sequence of the first heavy chain is as set forth in SEQ ID NO: 15, or is an amino acid sequence that is at least 80% identical to SEQ ID NO: 15.
  • the immunomodulatory agent is linked to the Fc region of the antibody that specifically binds a tumor antigen or immune checkpoint.
  • the first heavy chain comprises the constant region of an immunoglobulin selected from the group consisting of IgGl, IgG2, IgG3 and IgG4.
  • the first heavy chain contains 1 or more Fc regions of the same or different types fused to the immunomodulator through a polypeptide linker.
  • the immunomodulatory agent is linked to the N-terminus of the Fc region via a polypeptide linker.
  • the polypeptide linker is 5-30 amino acids.
  • the first heavy chain contains one or more immunomodulators of the same or different types fused to each other and to the Fc region.
  • the present invention also provides a method for preparing the above-mentioned heterodimer fusion protein.
  • the preparation method is to transfer three recombinant plasmids containing the above-mentioned light chain, first heavy chain and second heavy chain respectively into the same host cell. Recombinant expression.
  • the host cell is a mammalian cell, bacterial, fungal or insect cell.
  • the mammalian cell is a CHO cell, SP20 cell, NSO cell, COS cell, BHK cell, HEK293 cell or PerC6 cell.
  • the mammalian cells are CHO cells.
  • the present invention also provides a nucleic acid encoding the above-mentioned heterodimeric fusion protein.
  • the invention also provides a vector or plasmid containing the above nucleic acid.
  • the invention also provides a cell expressing the above vector or plasmid.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned heterodimeric fusion protein and at least one pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition can be used alone or in combination with other therapeutic agents to increase efficacy or reduce potential side effects.
  • the present invention also provides the application of the above-mentioned heterodimer fusion protein in preparing drugs for treating tumor diseases.
  • the neoplastic disease includes colorectal cancer, colorectal cancer, pancreatic adenocarcinoma, 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 sarcoma, nephroblastoma, neuroblastoma, ganglioneuroma, medulloblastoma, high-grade glaucoma
  • stromal tumors diffuse intrinsic pontine gliomas, and multilayered rosette embryonal tumors.
  • the present invention also provides the application of the above-mentioned heterodimeric fusion protein in preparing reagents or kits for detecting tumor antigens or immune checkpoints and IL-10 receptor molecules.
  • Figure 1 Schematic structural diagram of the heterodimeric fusion protein of the present invention.
  • Figure 2 ELISA detection of the binding activity of fusion proteins 1 and 2 to HER2 protein.
  • Figure 3 ELISA detection of the binding activity of fusion protein 2 to IL-10 receptor protein.
  • Figure 4 Binding activity of the heterodimeric fusion protein of the present invention to BT474 cells
  • Figure 5 Verification of IL-10 end luciferase expression activation of positive control and negative control.
  • Figure 6 Verification of luciferase expression activation at the IL-10 end of fusion protein 1.
  • Figure 7 Verification of luciferase expression activation at the IL-10 end of fusion protein 2.
  • heterodimer generally refers to a molecule (such as a protein molecule) consisting of two different members.
  • the two members of a heterodimer may differ in structure, function, activity and/or composition.
  • two different members may comprise polypeptides that differ in the order, number, or type of amino acid residues that form the polypeptides.
  • Each of the two different members of a heterodimer may independently comprise one, two or more units, polypeptide chains or moieties.
  • 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.
  • the targeting moiety may be an antibody, an antigen-binding antibody fragment, a bispecific antibody, or other antibody-based molecule or compound.
  • Other examples of targeting moieties may include, but are not limited to, aptamers, high-affinity multimers, receptor-binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides or proteins, and the like.
  • antigen binding site or "binding portion” generally refers to the portion of an antibody that participates in antigen binding.
  • the antigen binding site may be formed from the amino acid residues of the N-terminal variable ("V") region of the heavy (“H”) chain and/or light (“L”) chain.
  • V N-terminal variable
  • L light
  • Three highly divergent segments within the V regions of the heavy and light chains are called “hypervariable regions”, which are inserted between more conserved flanking segments called “framework regions” or "FRs” .
  • framework regions or "FRs”
  • the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged opposite each other in three-dimensional space to form an antigen-binding "surface.” This surface mediates recognition and binding of the target antigen.
  • 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 structural loop regions
  • the IMGT system defines CDRs based on sequence variability and position within the variable domain structure
  • AbM is based on Oxford Molecules Defined by the AbM antibody modeling software, it is a compromise between Kabat and Chothia
  • Contact defines CDRs based on the analysis of complex crystal structures, which is consistent with Chothia is similar.
  • the Kabat system is used for the numbering of amino acid positions (eg, amino acid residues in the Fc region) and target regions (eg, CDRs).
  • tumor antigen generally refers to an antigenic substance in or produced by tumor cells that may have the ability to trigger an immune response in the host.
  • a tumor antigen may be a protein, polypeptide, peptide or fragment thereof that forms part of a tumor cell and is capable of inducing tumor-specific cytotoxic T lymphocytes.
  • tumor antigen may also refer to an organism that is exclusively or preferentially or differentially expressed on cancer cells and/or is found to be associated with cancer cells thereby providing a target that is preferential or specific for cancer. Molecules (such as proteins, carbohydrates, glycoproteins, etc.).
  • preferential expression may be preferential expression compared to any other cell in the organism, or preferential expression within a specific region of the organism, such as within a specific organ or tissue.
  • inhibitory molecules usually refers to some inhibitory molecules and activating molecules present in the immune system, which can regulate the body's anti-tumor immune system by regulating T cell activity.
  • inhibitory molecules include PDL1, B7H3, CTLA4, etc.
  • activating molecules include OX40, 4-1BB, CD40, etc.
  • the term "immunomodulator” generally refers to substances that affect the function of the immune system. Immunomodulators can enhance or reduce immune responses.
  • the immunomodulatory agent may 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, Recombinant, synthetic and/natural preparations of interleukins, cytosine phosphate-guanosine (CpG) oligodeoxynucleotides and dextran.
  • the immunomodulatory agent is a cytokine.
  • polypeptide linker generally refers to a synthetic amino acid sequence that links or links two polypeptide sequences (eg, links two polypeptide domains). Peptide linkers can connect two amino acid sequences through peptide bonds. In some embodiments, the polypeptide linkers of the present application link an immunomodulatory agent to the Fc region.
  • antibody generally refers to a protein comprising one or more polypeptides essentially encoded by an immunoglobulin gene or immunoglobulin gene fragment.
  • Immunoglobulin genes can include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes.
  • light chains may 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.
  • Antibodies used in the present application may have structural units comprising tetramers.
  • Each tetramer can be composed of two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kD) and a “heavy” chain (approximately 50-70 kD).
  • the N-terminus of each member may define a variable region of approximately 100 to 110 or more amino acids, which 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 chain and heavy chain, respectively.
  • Antibodies may exist as intact immunoglobulins or as a number of well-characterized fragments generated by digestion with various peptidases or de novo expression.
  • antibody may also include antibody fragments produced by modifying the entire antibody or de novo synthesis using recombinant DNA methods, including but not limited to Fab'2, IgG, IgM, IgA, IgE, scFv, dAb, Nanobodies, single antibodies, and doublets. chain antibodies.
  • 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 variable heavy and variable light chains are linked together (either directly or through a peptide linker) to form a continuous polypeptide.
  • the antibodies and fragments of the present application are bispecific.
  • a bispecific antibody or fragment thereof has binding specificity for at least two different epitopes (eg, at least one of the at least two different epitopes is a tumor-associated antigen).
  • antibodies and fragments may also be heterologous antibodies, for example they may be or may comprise two or more antibodies or antibody-binding fragments (e.g., Fab) linked together, where each antibody or fragment has a different specificity.
  • identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a control polypeptide sequence after aligning the sequences and introducing gaps where necessary to obtain maximum percent sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill of the art, for example using publicly available computer software, such as BLAST software or the FASTA package.
  • the term "at least 80% identity” means that the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the control polypeptide sequence is more than 80%, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.
  • host cell generally includes a single cell, cell line, or cell culture that can be or has been the recipient of a subject's plasmid or vector, which contains a polynucleotide disclosed herein, or which expresses a heterologous dipeptide of the present application. Aggregate protein.
  • Host cells can include progeny of a single host cell. Due to natural, accidental or intentional mutations, the offspring may not necessarily be identical (either morphologically or in terms of total genomic DNA complement) to the original parent cell.
  • Host cells may include cells transfected in vitro with vectors disclosed herein.
  • the host cell may be a bacterial cell (eg, E. coli), yeast cell, or other eukaryotic cell, such as COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, or myeloma cells.
  • vector generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into and/or between host cells.
  • the term may include vectors used primarily for the insertion of DNA or RNA into a cell, vectors used primarily for the replication of DNA or RNA, and expression vectors used for the transcription and/or translation of DNA or RNA. Also included are vectors that provide more than one of the above functions.
  • An "expression vector” is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • treatment refers to a method of obtaining beneficial or desired results including, but not limited to, therapeutic benefits and/or prophylactic benefits.
  • Therapeutic benefit generally refers to the eradication or reduction of the severity of the underlying condition being treated. Additionally, by eradicating, reducing the severity, or reducing the incidence of one or more physical symptoms associated with the underlying condition such that improvement is observed in the subject (although the subject may still be suffering from the underlying condition) Therapeutic Benefits.
  • the compositions may be administered to subjects who are at risk of developing a particular disease, or who report one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.
  • in vivo generally refers to events that occur within the body of a subject.
  • in vitro generally refers to events that occur outside a subject's body.
  • in vitro assays include 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.
  • subject generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cattle, sheep, goats, rabbits, mice, rats, or monkeys.
  • fusion protein The amino acid sequences of the heterodimeric fusion protein (hereinafter referred to as "fusion protein"), IL-10 and its variants, and the control antibodies mentioned in the examples are shown in Table 1.
  • Table 1 The structures of fusion proteins 1 and 2 are shown in Figure 1. The only difference is that IL-10 in fusion protein 1 is natural and IL-10 in fusion protein 2 is mutated.
  • the PcDNA3.1 vector was used as a special vector for expressing the light chain and heavy chain of the fusion protein.
  • the PcDNA3.1 vector contains the promoter CMV Promoter used in the heavy chain, the eukaryotic selection marker G418 tag and the prokaryotic selection tag Ampicilline.
  • Gene synthesis is performed to obtain the nucleotide sequences of the first heavy chain, second heavy chain, and light chain encoding genes of the fusion protein expression (i.e., the target gene).
  • the vector and the target fragment are double-digested with HindIII and XhoI, and then recovered and passed through DNA ligase is used for enzymatic ligation, and E. coli competent cells DH5 ⁇ are transformed. Positive clones are selected and plasmid extraction and enzyme digestion verification are performed to obtain a recombinant plasmid containing the first heavy chain, second heavy chain, and light chain encoding genes of the fusion protein. .
  • the recombinant plasmid containing the above target genes was transformed into E. coli competent cells DH5 ⁇ , and the transformed bacteria were spread in a solution containing 100 ⁇ g/mL ampicillin.
  • Culture on the LB plate select plasmid clones and culture them in liquid LB medium, shake the bacteria at 260 rpm for 14 hours, extract the plasmid with an endotoxin-free plasmid extraction kit, dissolve it in sterile water, and measure the concentration with a nucleic acid protein quantifier.
  • ExpiCHO was cultured at 37°C, 8% CO 2 , and 100 rpm to a cell density of 6 ⁇ 10 6 cells/mL.
  • the transfection plasmid concentration is 1 mg/mL.
  • the liposome concentration is determined with reference to the ExpiCHO TM Expression System kit.
  • At 32°C , 5% CO 2 cultured at 100 rpm for 7-10 days. Feed once 18-22h after transfection and again on the 5th day. Place the above-mentioned culture product in a centrifuge and centrifuge at 4000g. Filter with a 0.22 ⁇ m filter and collect the culture supernatant.
  • Use ProteinA and an ion column to purify the resulting antibody protein and collect the eluate.
  • the specific steps for ProteinA and ion column purification are as follows: After high-speed centrifugation of the cell culture medium, take the supernatant and use GE's ProteinA chromatography column for affinity chromatography.
  • the equilibrium buffer used for chromatography is 1 ⁇ PBS (pH 7.4). After the cell supernatant is loaded and combined, it is washed with PBS until the UV light returns to the baseline. Then the target protein is eluted with the elution buffer 0.1M glycine (pH 3.0). Tris adjusts pH to neutral storage. Adjust the pH of the product obtained by affinity chromatography to 1-2 pH units below or above the isoelectric point pI, and dilute appropriately to control the sample conductivity below 5 ms/cm.
  • a 3-fold gradient dilution was performed for a total of 11 gradients, and a negative control (NEO-201 Antibody, msIgG1 subtype, see CN111670199A, heavy chain amino acid sequence is SEQ ID NO: 16, light chain amino acid sequence is SEQ ID NO: 17) and positive control antibody (INN trastuzumab, i.e. trastuzumab), 100 ⁇ L per well , incubate at 37°C for 1 hour.
  • NEO-201 Antibody msIgG1 subtype, see CN111670199A, heavy chain amino acid sequence is SEQ ID NO: 16, light chain amino acid sequence is SEQ ID NO: 17
  • positive control antibody INN trastuzumab, i.e. trastuzumab
  • the absorbance value of each well OD450nm-OD570nm.
  • the logarithm of the concentration of the fusion protein was taken as the abscissa, and the measured absorbance value of each well was used as the ordinate.
  • the Sigmoidal dose-response (Variable Slope) method (Graph Pad Prism Software, Graph Pad Software, SanDiego, California) was used for non-linear analysis. Linear regression was used to obtain the binding curve of the fusion protein and HER2 protein.
  • Fusion protein 2 can bind to HER2 in multiple concentration ranges.
  • Fusion protein 2 can bind to the IL-10 receptor in multiple concentration ranges, which is better than the positive control.
  • BT474 cells from Shanghai Chinese Academy of Sciences
  • FACS buffer at a density of 1 ⁇ 10 6 /mL.
  • the purified fusion protein was diluted to 200nM with FACS buffer. Using this as the starting concentration, a 5-fold gradient dilution was performed for a total of 8 gradients, and a negative control (i.e.
  • NEO-201 antibody msIgG1 subtype see CN 111670199A
  • the heavy chain amino acid sequence is SEQ ID NO: 16
  • the light chain amino acid sequence is SEQ ID NO: 17
  • the positive control INN trastuzumab
  • 100 ⁇ L of fusion protein diluent was added.
  • Cells were incubated at 4°C for 60 min and then washed twice with excess FACS buffer.
  • Cells were fixed in fixation buffer and subsequently analyzed by flow cytometry. FACS method was used to detect the binding activity of fusion protein and BT474 cells.
  • Fusion proteins 1 and 2 can specifically bind to BT474 in multiple concentration ranges.
  • IL-10 combines with IL-10R to mediate control of the degree and duration of inflammation, which is crucial for maintaining the body's inflammatory response homeostasis. This process relies on the regulation of signal transducer and activator of transcription 3 (STAT3).
  • STAT3 signal transducer and activator of transcription 3
  • This experiment uses HEK293 tool cells, which stably express the IL-10R and STAT3 signaling pathway reporter gene system, and uses IL-10 protein stimulation to activate the increase in cell luciferase expression. Therefore this cell line was used for the activity study of IL-10-STAT3.
  • IL-10-Reporter-HEK-293 cell line purchased from Jiman Biotechnology
  • WHB-96-01 96-well cell culture plate
  • the positive control (the 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 respectively Use diluent (DMEM complete medium) to perform 4-fold gradient dilution starting from 300 ⁇ M, for a total of 9 concentration gradients, and add 100 ⁇ L/well to the 96-well cell culture plate from which the supernatant has been aspirated. After mixing, place it in a 37°C, 5% CO2 incubator and incubate for 6 hours.
  • DMEM complete medium 4-fold gradient dilution starting from 300 ⁇ M, for a total of 9 concentration gradients
  • Bio-Lite Luciferase Assay substrat (Vazyme, Cat: DD1201-02) chromogenic solution, 100 ⁇ L/well, and incubate at room temperature for 10 minutes. Then use a microplate reader to read and detect the fluorescence value.
  • the EC 50 of the positive control and fusion protein 1 are 2.259nM and 5.839nM respectively, and the relative activity value of the two is 39%.
  • the EC 50 of the positive control and fusion protein 2 are 2.117nM and 0.648nM respectively, and the relative activity value of the two is 327%. It can be seen that the activation activity of fusion protein 2 is better than that of fusion protein 1.

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Abstract

一种异源二聚体融合蛋白及其应用。该异源二聚体融合蛋白包含：第一重链，所述第一重链包含Fc区域、与Fc区域融合的免疫调节剂；轻链和第二重链，所述轻链和第二重链复合以形成表现出对肿瘤抗原或免疫检查点的结合特异性的靶向部分；所述轻链、第一重链、第二重链复合以形成所述异源二聚体融合蛋白，其中所述第一重链中的免疫调节剂可以含有突变。该异源二聚体融合蛋白对肿瘤相关靶向抗原和IL-10受体均有较高的亲和力，并且具有良好的抗肿瘤活性。

Description

一种异源二聚体融合蛋白及其应用 技术领域
本发明属于生物医药技术领域，具体涉及一种异源二聚体融合蛋白及其应用。
背景技术
IL-10主要由激活的T细胞和抗原提呈细胞分泌，抗原识别的过程中，CD8+T细胞中的IL-10受体(IL-10R)表达上调。IL-10由特异的细胞表面受体复合物介导多种活性，IL-10受体包含两个不同的链，IL-10R1和IL-10R2，两条链均属于II类细胞因子受体家族(CRF2)。在细菌感染和组织损伤中，IL10可以减少炎症反应，抑制T细胞(Th17)和巨噬细胞引起的炎症反应(IL-12/23)，降低肿瘤相关炎症反应。在肿瘤微环境中，IL-10可以高效激活抗原特异性CD8+T细胞的增殖和毒性。
IL-10抗肿瘤的作用机制为：a.可以激活肿瘤内部CD8+T细胞的活性和扩增；b.IL10可以增加肿瘤内部抗原特异性T淋巴细胞的活性和扩增；c.IL-10对肿瘤的排斥作用具有记忆功能，动物体内试验数据表明，给予IL-10药物肿瘤消失后，再次给予小鼠接种肿瘤细胞，该肿瘤细胞不在小鼠体内生长，主要的原因为：IL-10能够增强抗原特异性CD8+T细胞的存活率，起到肿瘤疫苗的作用；d.IL10通过恢复终端耗竭T细胞的氧化磷酸化代谢来重新激活T细胞达到杀伤肿瘤细胞的目的。临床试验也证明，与PDL1抗体联合使用时，会增加肿瘤内部细胞PDL1特异性CD8+阳性细胞，产生持久的抗肿瘤效果。但目前暂未有以IL-10为靶点的上市药物。IL-10可以促进针对特异性抗原的CD8+T细胞的扩增和存活期，而特异性抗原CD8+T细胞与免疫细胞对肿瘤的杀伤呈正相关性。虽然已有多项研究表明，免疫调节剂可用于在动物模型和癌症患者中施加抗肿瘤作用，然而，与免疫调节剂的应用相关的短半衰期和系统性毒性极大地限制了它们的使用。IL-10与受体结合以后，启动STAT3、STAT1通路，这两条通路是IL-10起生物功能的信号转导途径。
专利CN201380041222.1中提供包含未突变的IL-10的融合蛋白。然而，这种融合蛋白的表达量低，纯度不高，IL-10端的结合活性与信号激活能力弱，与癌细胞结合活性差，无法用于规模化工业生产。因此，有必要寻找既与IL-10具有良好结合活性，又与肿瘤相关抗原具有良好结合活性的蛋白。
发明内容
本发明目的在于提供一种异源二聚体融合蛋白及其应用。该异源二聚体融合蛋白对肿瘤抗原或免疫检查点和IL-10受体均有较高的亲和力，且具有良好的抗肿瘤活性。
本发明提供一种异源二聚体融合蛋白，所述异源二聚体融合蛋白包含：第一重链，所述第一重链包含Fc区域、与Fc区域融合的免疫调节剂；轻链和第二重链，所述轻链和第二重链复合以形成表现出对肿瘤抗原或免疫检查点的结合特异性的靶向部分；所述轻链、第一重链、第二重链复合以形成所述异源二聚体融合蛋白，其中所述第一重链中的免疫调节剂可以含有突变。
在一个实施方案中，免疫调节剂为细胞因子、细胞因子受体、生长因子、激素或细胞外基质分子。在一个实施方案中，所述免疫调节剂选自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-12Rα、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、IL-31R中的一种或多种。在一个实施方案中，所述免疫调节剂为IL-10。
在一个实施方案中，所述IL-10的氨基酸序列如SEQ ID NO:1或2所示，或为与SEQ ID NO:1或2具有至少80％同一性的氨基酸序列。
在一个实施方案中，所述IL-10的氨基酸序列如SEQ ID NO:1所示，其可以包含如下突变位点中的一个或多个：N18Y、R104W、N92Q、T100D。
在一个实施方案中，肿瘤抗原或免疫检查点为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、TL1A、MART-1/MelanA、gp100、酪氨酸酶、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、爱泼斯坦巴尔病毒抗原EBVA、人类乳头瘤病毒抗原E6或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-连环蛋白、CDK4、Mum-1、p 15、p 16、43-9F、5T4、791Tgp72、甲胎蛋白、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结合蛋白\亲环蛋白C-相关蛋白、TAAL6、TAG72、TLP、MUC16、IL13Rα2、FRα、VEGFR2、Lewis Y、FAP、EphA2、CEACAM5、CEACAM6、EGFR、CA6、CA9、GPNMB、EGP1、FOLR1、内皮受体、STEAP1、SLC44A4、结合素-4、 AGS-16、胍基环化酶C、MUC-1、CFC1B、整联蛋白α3链、TPS、CD19、CD20、CD22、CD30、CD72、CD180、CD171、CD123、CD133、CD138、CD37、CD70、CD79a、CD79b、CD56、CD74、CD166、CD71、CLL-1/CLEC12A、ROR1、磷脂酰肌醇蛋白聚糖3、间皮素、CD33/IL3Ra、c-Met、PSCA、PSMA、糖脂F77、EGFRvIII、BCMA、GD-2、MY-ESO-1或MAGE A3中的一种或多种。
在一个实施方案中，所述轻链包含互补决定区(CDR)，所述互补决定区包含与特异性结合肿瘤抗原或免疫检查点的抗体的轻链相应CDR的氨基酸序列具有至少80％同一性的氨基酸序列。
在一个实施方案中，特异性结合肿瘤抗原或免疫检查点的抗体的轻链含氨基酸序列如SEQ ID NO:6所示的LCDR1、氨基酸序列如SEQ ID NO:7所示的LCDR2、氨基酸序列如SEQ ID NO:8所示的LCDR3。
在一个实施方案中，所述轻链包含可变区，所述可变区包含与特异性针对肿瘤抗原或免疫检查点的抗体的轻链可变区中包含的氨基酸序列具有至少80％同一性的氨基酸序列。
在一个实施方案中，所述轻链的可变区的氨基酸序列如SEQ ID NO:10所示，或为与SEQ ID NO:10具有至少80％同一性的氨基酸序列。
在一个实施方案中，所述轻链的氨基酸序列如SEQ ID NO:13所示，或为与SEQ ID NO:13具有至少80％同一性的氨基酸序列。
在一个实施方案中，所述第二重链包含互补决定区(CDR)，所述互补决定区包含与特异性结合肿瘤抗原或免疫检查点的抗体的第二重链相应CDR的氨基酸序列具有至少80％同一性的氨基酸序列。
在一个实施方案中，所述特异性结合肿瘤抗原或免疫检查点的抗体的第二重链含氨基酸序列如SEQ ID NO:3所示的HCDR1、氨基酸序列如SEQ ID NO:4所示的HCDR2、氨基酸序列如SEQ ID NO:5所示的HCDR3。
在一个实施方案中，所述第二重链包含可变区，所述可变区包含与特异性针对肿瘤抗原或免疫检查点的抗体的轻链可变区中包含的氨基酸序列具有至少80％同一性的氨基酸序列。
在一个实施方案中，所述第二重链的可变区氨基酸序列如SEQ ID NO:9所示，或为与SEQ ID NO:9具有至少80％同一性的氨基酸序列。
在一个实施方案中，所述第二重链的氨基酸序列如SEQ ID NO:12所示，或为与SEQ ID NO:12具有至少80％同一性的氨基酸序列。
在一个实施方案中，所述第一重链的氨基酸序列如SEQ ID NO:15所示，或为与SEQ ID NO:15具有至少80％同一性的氨基酸序列。
在一个实施方案中，所述免疫调节剂与所述特异性结合肿瘤抗原或免疫检查点的抗体的Fc区域连接。
在一个实施方案中，所述第一重链包含选自IgG1、IgG2、IgG3和IgG4的免疫球蛋白的恒定区。
在一个实施方案中，所述第一重链含1个或更多个相同或不同类型的Fc区域，所述Fc区域通过多肽接头与所述免疫调节剂融合。在一个实施方案中，所述免疫调节剂通过多肽接头连接至Fc区域的N端。在一个实施方案中，所述多肽接头为5-30个氨基酸。在一个实施方案中，所述多肽接头为(GGGGS)n，其中n＝1-6。
在一个实施方案中，所述第一重链含一个或多个相同或不同类型的免疫调节剂，所述一个或多个免疫调节剂相互融合且与所述Fc区域融合。
本发明还提供了上述异源二聚体融合蛋白的制备方法，所述制备方法是将分别含上述轻链、第一重链、第二重链的三个重组质粒转入同一宿主细胞中进行重组表达。在一个实施方案中，所述宿主细胞为哺乳动物细胞、细菌、真菌或昆虫细胞。在一个实施方案中，所述哺乳动物细胞为CHO细胞、SP20细胞、NSO细胞、COS细胞、BHK细胞、HEK293细胞或PerC6细胞。在一个实施方案中，所述哺乳动物细胞为CHO细胞。
本发明还提供了一种编码上述异源二聚体融合蛋白的核酸。本发明还提供了一种含上述核酸的载体或质粒。本发明还提供了一种表达上述载体或质粒的细胞。
本发明还提供了一种药物组合物，所述药物组合物包含上述的异源二聚体融合蛋白以及至少一种药学上可接受的赋形剂、稀释剂或载体。在一个实施方案中，所述药物组合物可以单独使用或与其他治疗剂联用，来提高疗效或减少潜在的副作用。
本发明还提供了上述异源二聚体融合蛋白在制备治疗肿瘤疾病的药物方面的应用。在一个实施方案中，所述肿瘤疾病包括大肠癌、结肠直肠癌、膜腺癌、肺癌、食管癌、前列腺癌、促结缔组织增生性小圆细胞肿瘤、卵巢癌、胃癌、胰腺癌、肝癌、肾癌、乳腺癌、非小细胞肺癌、黑色素瘤、肺泡横纹肌肉瘤、胚胎性横纹肌肉瘤、尤因肉瘤、肾母细胞瘤、神经母细胞瘤、神经节细胞瘤、髓母细胞瘤、高级别胶质瘤、弥漫性内在脑桥胶质瘤、多层菊形团胚胎性肿瘤中的一种或多种。
本发明还提供了上述异源二聚体融合蛋白在制备检测肿瘤抗原或免疫检查点和IL-10受体分子的试剂或试剂盒方面的应用。
附图说明
图1：本发明的异源二聚体融合蛋白的结构示意图。
图2：ELISA检测融合蛋白1和2对HER2蛋白的结合活性。
图3：ELISA检测融合蛋白2对IL-10受体蛋白的结合活性。
图4：本发明的异源二聚体融合蛋白与BT474细胞结合活性图5：阳性对照和阴性对照的IL-10端荧光素酶表达激活验证。
图6：融合蛋白1的IL-10端荧光素酶表达激活验证。
图7：融合蛋白2的IL-10端荧光素酶表达激活验证。
具体实施方式
下面结合实施例对本发明作进一步说明，实施例用于描述本发明的一些具体实施方案，而非用于限制本发明的保护范围。除非另有定义，否则本文使用的所有技术和科学术语具有与本申请所属领域的普通技术人员通常理解的相同的含义。虽然与本文所述的方法和材料相似或等同的方法和材料可用于本申请的实践或测试，但下文描述了合适的方法和材料。在矛盾的情况下，以专利说明书为准。
术语“异源二聚体”通常是指由两个不同成员组成的分子(例如蛋白质分子)。异源二聚体的两个成员可在结构、功能、活性和/或组成方面不同。例如，两种不同的成员可以包含在形成这些多肽的氨基酸残基的顺序、数目或种类上不同的多肽。异源二聚体的两个不同成员中的每一个可以独立地包含一个、两个或更多个单元、多肽链或部分。
术语“靶向部分”通常是指与靶分子、细胞、颗粒、组织或聚集体特异性、选择性或优先结合的分子、复合物或聚集体。例如，靶向部分可以是抗体、抗原结合性抗体片段、双特异性抗体或其他基于抗体的分子或化合物。靶向部分的其他实例可以包括但不限于适体、高亲和性多聚体、受体结合性配体、核酸、生物素-亲和素结合对、结合肽或蛋白质等。
术语“抗原结合位点”或“结合部分”通常是指参与抗原结合的抗体的一部分。抗原结合位点可以由重(“H”)链和/或轻(“L”)链的N-末端可变(“V”)区的氨基酸残基形成。重链和轻链的V区内的三个高度趋异的区段被称为“高变区”，其被插入在称为“框架区”或“FR”的更保守的侧翼区段之间。在抗体分子中，轻链的三个高变区和重链的三个高变区在三维空间中彼此相对地排列，以形成抗原结合“表面”。该表面可介导所述靶抗原的识别和结合。
本领域中有多种方法/系统来定义和描述CDR，这些系统和/或定义已经开发和精制多年，包括Kabat、Chothia、IMGT、AbM和Contact。Kabat是最常用的，基于序列变异性定义CDR；Chothia基于结构循环区域的位置基于序列变异性定义CDR；IMGT系统基于可变域结构内的序列变异性和位置定义CDR；AbM是基于牛津分子公司的AbM抗体建模软件进行定义，是Kabat和Chothia之间的折衷；Contact基于对复杂晶体结构的分析定义CDR，在多个方面与 Chothia类似。本发明中氨基酸位置的编号(例如Fc区的氨基酸残基)和目标区域(例如CDR)，使用Kabat系统。
术语“肿瘤抗原”通常是指在肿瘤细胞中或由肿瘤细胞产生的抗原物质，其可具有在宿主中触发免疫应答的能力。例如，肿瘤抗原可以是构成肿瘤细胞的一部分并且能够诱导肿瘤特异性细胞毒性T淋巴细胞的蛋白质、多肽、肽或其片段。在一些实施方案中，术语“肿瘤抗原”还可指在癌细胞上唯一地或优先地或差异地表达和/或经发现与癌细胞相关从而提供对癌症是优先的或特异性的靶标的生物分子(例如蛋白质、碳水化合物、糖蛋白等)。例如，优先表达可以是相较于生物体中的任何其他细胞的优先表达，或者在生物体的特定区域内(例如在特定器官或组织内)的优先表达。
术语“免疫检查点”通常指免疫系统中存在的一些抑制型分子和激活型分子，可通过调控T细胞活性，调控机体的抗肿瘤免疫体系。例如，抑制型分子包括PDL1、B7H3、CTLA4等，激活型分子包括OX40、4-1BB、CD40等。
术语“免疫调节剂”通常指影响免疫系统功能的物质。免疫调节剂可以增强或减小免疫应答。例如，免疫调节剂可以是免疫疗法的活性剂，包括但不限于例如细胞因子、粒细胞集落刺激因子(G-CSF)、干扰素、咪喹莫特、来自细菌的细胞膜片段、趋化因子、白细胞介素、胞嘧啶磷酸-鸟苷(CpG)寡脱氧核苷酸和葡聚糖的重组、合成和/天然制剂。在一些实施方案中，所述免疫调节剂是细胞因子。
术语“多肽接头”通常是指连接或联接两个多肽序列(例如联接两个多肽结构域)的合成氨基酸序列。多肽接头可以通过肽键连接两个氨基酸序列。在一些实施方案中，本申请的多肽接头将免疫调节剂连接至Fc区域。
术语“抗体”通常是指包含一个或多个基本上由免疫球蛋白基因或免疫球蛋白基因片段编码的多肽的蛋白质。免疫球蛋白基因可以包括κ、λ、α、γ、δ、ε和μ恒定区基因，以及无数的免疫球蛋白可变区基因。如本文所用，轻链可被分类为κ或λ。重链可被分类为γ、μ、α、δ或ε，其依次分别定义免疫球蛋白类别：IgG、IgM、IgA、IgD和IgE。本申请中使用的抗体可具有包含四聚体的结构单元。每个四聚体可由两对相同的多肽链组成，每对具有一条“轻”链(约25kD)和一条“重链”(约50-70kD)。每个成员的N末端可以界定约100至110个或更多个氨基酸的可变区，其主要负责抗原识别。术语轻链可变区(VL)和重链可变区(VH)通常分别指轻链和重链的这些区域。抗体可作为完整免疫球蛋白存在或作为通过用各种肽酶消化或从头表达产生的许多充分表征的片段存在。术语“抗体”还可包括通过修饰整个抗体或使用重组DNA方法从头合成产生的抗体片段，包括但不限于Fab'2、IgG、IgM、IgA、IgE、scFv、dAb、纳米抗体、单抗体和双链抗体。在一些实施方案中，抗体包括但不限于Fab'2、IgG、IgM、IgA、 IgE和单链抗体，例如单链Fv(scFv)抗体，其中可变重链和可变轻链(直接地或通过肽接头)连接在一起以形成连续的多肽。在一些实施方案中，本申请中的抗体和片段是双特异性的。在一些实施方案中，双特异性抗体或其片段对至少两个不同表位(例如，至少两个不同表位中的至少一个是肿瘤相关抗原)具有结合特异性。在一些实施方案中，抗体和片段也可以是异种抗体，例如它们可以是或可以包含两个或更多个连接在一起的抗体或抗体结合片段(例如Fab)，其中每个抗体或片段具有不同的特异性。
术语“同一性”定义为比对序列并在必要时引入缺口以获取最大百分比序列同一性后，候选序列中与对照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式进行，例如使用公众可得到的计算机软件，诸如BLAST软件或FASTA程序包。
术语“至少80％同一性”是指候选序列中与对照多肽序列中的氨基酸残基相同的氨基酸残基的百分率为80％以上，包括80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％。
术语“宿主细胞”通常包括可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物，其包含本申请公开的多核苷酸，或表达本申请的异源二聚体蛋白质。宿主细胞可以包括单个宿主细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始亲本细胞完全相同(在形态上或在基因组总DNA互补体上)。宿主细胞可包括用本申请公开的载体在体外转染的细胞。宿主细胞可以是细菌细胞(例如大肠杆菌(E.coli))、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、HeLa细胞或骨髓瘤细胞。
术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移至宿主细胞中和/或在宿主细胞之间转移。该术语可包括主要用于将DNA或RNA插入细胞的载体，主要用于DNA或RNA的复制的载体，以及用于DNA或RNA的转录和/或翻译的表达载体。还包括提供不止一种上述功能的载体。“表达载体”是当被引入合适的宿主细胞时可被转录并翻译成多肽的多核苷酸。
术语“治疗”是指获得有益或所需的结果(包括但不限于治疗益处和/或预防益处)的方法。治疗益处通常是指根除或减轻所治疗的潜在病症的严重性。此外，通过根除、减轻严重性或减少与潜在病症相关的一种或多种生理症状的发生率，以使得在受试者中观察到改善(尽管受试者仍然可能受到潜在病症折磨)来实现治疗益处。对于预防益处，可向处于发展特定疾病的风险中的受试者，或报告疾病的一种或多种生理症状的受试者施用组合物，即使可能尚未进行该疾病的诊断。
术语“体内”通常是指在受试者体内发生的事件。
术语“体外”通常是指发生在受试者体外的事件。例如，体外测定包括在受试者外进行的任何测定。体外测定包括其中使用死细胞或活细胞的基于细胞的测定。体外测定还包括其中不使用完整细胞的无细胞测定。
术语“受试者”通常是指人或非人动物，包括但不限于猫、狗、马、猪、牛、绵羊、山羊、兔、小鼠、大鼠或猴。
本文中的异源二聚体融合蛋白(以下简称“融合蛋白”)，IL-10及其变体，以及实施例中提及到的对照抗体的氨基酸序列如表1所示。融合蛋白1和2结构如图1所示，区别仅在于融合蛋白1中IL-10为天然的，融合蛋白2中IL-10为突变的。
表1.本发明的融合蛋白、IL10等的氨基酸序列




实施例1基因合成与表达载体的构建
采用PcDNA3.1载体作为表达融合蛋白的轻链和重链的专用载体。PcDNA3.1载体含有重链所使用的启动子CMV Promoter、真核筛选标记G418标签和原核筛选标签Ampicilline。基因合成分别得到融合蛋白表达的第一重链、第二重链、轻链编码基因的核苷酸序列(即目的基因)，用HindIII和XhoI对载体和目的片段进行双酶切，回收后通过DNA连接酶进行酶连，并转化大肠杆菌感受态细胞DH5α，挑选出阳性克隆并进行质粒提取和酶切验证，获得含融合蛋白第一重链、第二重链、轻链编码基因的重组质粒。
实施例2质粒抽提
根据《分子克隆实验指南》(2002年，科学出版社)所述方法，将含有上述各目的基因的重组质粒转化至大肠杆菌感受态细胞DH5α中，将转化细菌涂布在含100μg/mL氨苄青霉素的LB平板上培养，挑选质粒克隆至液体LB培养基中培养，260rpm摇菌14h，由无内毒素质粒大抽试剂盒抽提质粒，用无菌水溶解并用核酸蛋白定量仪进行浓度测定。
实施例3质粒转染、瞬转表达与融合蛋白纯化
在37℃、8％CO2、100rpm下培养ExpiCHO至细胞密度6×106个/mL。使用脂质体分别将构建的载体质粒按照质量浓度1:1:1转染到上述细胞中，转染质粒浓度为1mg/mL，脂质体浓度参照ExpiCHOTM Expression System试剂盒确定，在32℃、5％CO2，100rpm下培养7-10天。转染18-22h之后和第5天之间分别补料一次。将上述培养产物置于离心机中，以4000g转速离心，0.22μm滤膜过滤并收集培养基上清液，采用ProteinA、离子柱纯化所得的抗体蛋白并收集洗脱液。
ProteinA、离子柱纯化的具体操作步骤为：细胞培养液经过高速离心后取上清，利用GE的ProteinA层析柱进行亲和层析。层析使用平衡缓冲液为1×PBS(pH 7.4)，细胞上清上样结合后利用PBS洗涤至紫外线回到基线，然后利用洗脱缓冲液0.1M甘氨酸(pH 3.0)洗脱目的蛋白，利用Tris调节pH至中性保存。将亲和层析所得产物调节pH至低于或者高于等电点pI1-2个pH单位，适当稀释以控制样本电导在5ms/cm以下。利用合适的对应pH缓冲液如磷酸缓冲液、醋酸缓冲液等条件，利用本领域内常规的离子交换层析方法如阴离子交换或者阳离子交换进行对应pH条件下NaCl梯度洗脱，根据SDS-PAGE选择目的蛋白所在的收集管合并保存。然后，将纯化后所得的洗脱液超滤换液至缓冲液中。
实施例4 ELISA检测融合蛋白对HER2的亲和力
采用pH 7.4的PBS缓冲液将huHER2-his(购于ACRO,CAT:HE2-H5225)稀释至0.5μg/mL，每孔100μL加入到96孔ELISA板中，4℃包被过夜。用1％BSA封闭液封闭1小时后。PBST洗板3次后，将纯化得到的融合蛋白用0.5％BSA样品稀释液稀释至100nM，以此为起始浓度，进行3倍梯度稀释，共11个梯度，并设阴性对照(NEO-201抗体，msIgG1亚型，参见CN111670199A，重链氨基酸序列为SEQ ID NO：16，轻链氨基酸序列为SEQ ID NO：17)与阳性对照抗体(INN trastuzumab，即曲妥珠单抗)，每孔100μL，37℃孵育1h。再用PBST洗板3次，将HRP标记的山羊抗人IgG Fc(Jackson Cat:109-035-098)用样品稀释液按1:10000稀释，每孔加入100μL，室温孵育1h。PBST洗板4次后，每孔加入100μL TMB底物，室温避光孵育10min，每孔加入100μL 1M HCl液终止显色反应。在多功能酶标仪上选择波长450nm， 参比波长570nm测定96孔板中各孔的吸光值，每孔吸光值(OD)＝OD450nm-OD570nm。将融合蛋白的浓度取对数后作为横坐标，测得的每孔吸光值为纵坐标，选用Sigmoidal dose-response(Variable Slope)方式(Graph Pad Prism软件，Graph Pad Software，SanDiego，California)进行非线性回归，得到融合蛋白与HER2蛋白的结合曲线。
融合蛋白的ELISA结果如图2所示，融合蛋白2在多浓度范围下可与HER2结合。
实施例5 ELISA检测融合蛋白对IL-10受体的亲和力
采用pH 7.4的PBS缓冲液将IL-10受体人IL10RA-his(购于北京义翘神州,CAT:10419-H08H)稀释至0.8μg/mL，每孔100μL加入到96孔ELISA板中，4℃包被过夜。用1％BSA封闭液封闭1小时。PBST洗板3次后，将纯化得到的抗体用0.5％BSA样品稀释液稀释至100nM，以此为起始浓度，进行3倍梯度稀释，共11个梯度，并设阴性对照(INN trastuzumab，即曲妥珠单抗)与阳性对照(专利202110141918.8实施例4中得到的融合蛋白)，每孔100μL，37℃孵育1h。再用PBST洗板3次，将HRP标记的山羊抗人IgG Fc(Jackson Cat:109-035-098)用样品稀释液按1:10000稀释，每孔加入100μL，室温孵育1h。PBST洗板4次后，每孔加入100μL TMB底物，室温避光孵育10min，每孔加入100μL 1M HCl液终止显色反应。
在多功能酶标仪上选择波长450nm，参比波长570nm测定96孔板中各孔的吸光值，每孔吸光值(OD)＝OD450nm-OD570nm。将抗体的浓度取对数后作为横坐标，测得的每孔吸光值为纵坐标，选用Sigmoidal dose-response(Variable Slope)方式(Graph Pad Prism软件，Graph Pad Software，SanDiego，California)进行非线性回归，得到目标融合蛋白与IL-10受体IL-10RA蛋白的结合曲线。
融合蛋白的ELISA结果如图3所示，融合蛋白2在多浓度范围下均可与IL-10受体结合，优于阳性对照。
实施例6融合蛋白的IL-10生物活性HER2端与BT474细胞的结合活性
取对数期生长、形态正常的BT474细胞(来自上海中科院)，转至离心管1000rpm离心5min，按照1×106/mL的密度使用FACS缓冲液重悬细胞，取100uL细胞分至独立的管中。将纯化得到的融合蛋白用FACS缓冲液稀释至200nM，以此为起始浓度，进行5倍梯度稀释，共8个梯度，并设阴性对照(即NEO-201抗体msIgG1亚型，参见CN 111670199A，重链氨基酸序列为SEQ ID NO：16，轻链氨基酸序列为SEQ ID NO：17)与阳性对照(INN trastuzumab)，并添加100μL融合蛋白稀释液。将细胞在4℃下孵育60分钟，然后用过量FACS缓冲液洗涤两次。将细胞重新悬浮于100μL FACS缓冲液中，并将羊抗人二抗-PE(Biolgend，Cat:398004)) 添加到样品中，孵育30分钟并用过量FACS缓冲液洗涤两次。将细胞在固定缓冲液中固定，随后通过流式细胞术进行分析。FACS方法检测融合蛋白与BT474细胞结合活性。
融合蛋白1和2与BT474细胞结合活性的FACS检测结果如图4所示，融合蛋白1和2在多浓度范围下均可与BT474特异结合。
实施例7 IL-10端荧光素酶表达激活验证
IL-10与IL-10R结合，介导控制炎症的程度和炎症的持续时间，这对于维持机体炎症反应稳态至关重要。这一过程依赖于信号转导和转录激活因子3(STAT3)的调控。该实验使用HEK293工具细胞，稳定表达IL-10R及STAT3信号通路报告基因系统，使用IL-10蛋白刺激，能够激活细胞荧光素酶表达上升。因此使用此细胞系用于IL-10-STAT3的活性研究。
取对数生长期的IL-10-Reporter-HEK-293细胞株(购于吉满生物)，每孔2.5×104个细胞铺于96孔细胞培养板(WHB，Cat:WHB-96-01)，于37℃、5％CO2培养箱中过夜培养。之后阳性对照(专利202110141918.8实施例4中得到的融合蛋白)、IL-10蛋白(Novoprotein，Cat：CX04)、阴性对照(西妥昔单抗)、人-IgG1、融合蛋白1、融合蛋白2分别用稀释液(DMEM完全培养基)分别从300μM起进行4倍梯度稀释，共9个浓度梯度，100μL/孔加入吸除上清的96孔细胞培养板中。混匀后置于37℃、5％CO2培养箱中孵育6h。而后加入Bio-Lite Luciferase Assay substrat(Vazyme，Cat：DD1201-02)显色液，100μL/孔，常温孵育10min。之后用酶标仪进行读数，检测荧光值。
以融合蛋白浓度为横坐标，相对发光值(RLU)为纵坐标，采用GraphPad Prism软件Graphpad Prism 5 Demo，San Diego，California)中非线性回归分析方法，选择Log(agonist)v.s.Response--Variable Slope方式做拟合曲线图，得到融合蛋白荧光量效曲线。
如图5所示，IL-10与阳性对照呈剂量依赖性地激活HEK293细胞荧光素酶表达，而阴性对照西妥昔单抗与人IgG1无此效果，表示此报告基因方法可反应IL-10的信号激活活性。计算阳性对照EC50/融合蛋白EC50，如果该相对活性值大于100％，说明融合蛋白的活性优于阳性对照，反之，融合蛋白的活性低于阳性对照。图6中，阳性对照和融合蛋白1的EC50分别为2.259nM和5.839nM，二者的相对活性值为39％。图7中，阳性对照和融合蛋白2的EC50分别为2.117nM和0.648nM，二者的相对活性值为327％。可见，融合蛋白2的激活活性优于融合蛋白1。
应当说明的是，以上所述仅为本发明的较佳实施例而已，并不用于限制本发明的范围，凡在本发明的精神和原则之内所作出的任何修改、等同的替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种异源二聚体融合蛋白，其特征在于，所述异源二聚体融合蛋白包含：第一重链，所述第一重链包含Fc区域、与Fc区域融合的免疫调节剂；轻链和第二重链，所述轻链和第二重链复合以形成表现出对肿瘤抗原或免疫检查点的结合特异性的靶向部分；所述轻链、第一重链、第二重链复合以形成所述异源二聚体融合蛋白，其中所述第一重链中的免疫调节剂可以含有突变。
  2. 根据权利要求1所述的异源二聚体融合蛋白，其特征在于，所述免疫调节剂为细胞因子、细胞因子受体、生长因子、激素或细胞外基质分子；优选地，所述免疫调节剂选自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中的一种或多种；更优选地，所述免疫调节剂为IL-10。
  3. 根据权利要求1或2所述的异源二聚体融合蛋白，其特征在于，所述免疫调节剂包含至少1-4个突变。
  4. 根据权利要求3所述的异源二聚体融合蛋白，其特征在于，该突变的位点包含N18Y、R104W、N92Q、T100D中的一个或多个。
  5. 根据权利要求1-4中任一项所述的异源二聚体融合蛋白，其特征在于，所述第一重链含一个或多个免疫调节剂，所述免疫调节剂相互融合且与所述Fc区域融合；优选地，所述免疫调节剂是IL-10；更优选地，所述第一重链的氨基酸序列如SEQ ID NO:15所示，或为与SEQ ID NO:15具有至少80％同一性的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的异源二聚体融合蛋白，其特征在于，所述肿瘤抗原或免疫检查点为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、TL1A、MART-1/MelanA、gp100、酪氨酸酶、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、爱泼斯坦巴尔病毒抗原EBVA、人类乳头瘤病毒抗原E6或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-连环蛋白、CDK4、Mum-1、p 15、p 16、43-9F、5T4、791Tgp72、甲胎蛋白、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结合蛋白\亲环蛋白C-相关蛋白、TAAL6、TAG72、TLP、MUC16、IL13Rα2、FRα、VEGFR2、Lewis Y、FAP、EphA2、CEACAM5、CEACAM6、EGFR、CA6、CA9、GPNMB、EGP1、FOLR1、内皮受体、STEAP1、SLC44A4、结合素-4、AGS-16、胍基环化酶C、MUC-1、CFC1B、整联蛋白α3链、TPS、CD19、CD20、CD22、CD30、CD72、CD180、CD171、CD123、CD133、CD138、CD37、CD70、CD79a、CD79b、CD56、CD74、CD166、CD71、CLL-1/CLEC12A、ROR1、磷脂酰肌醇蛋白聚糖3、间皮素、CD33/IL3Ra、c-Met、PSCA、PSMA、糖脂F77、EGFRvIII、BCMA、GD-2、MY-ESO-1或MAGE A3中的一种或多种。
  7. 根据权利要求6所述的异源二聚体融合蛋白，其特征在于，所述轻链和第二重链均包含互补决定区，所述互补决定区包含与特异性结合肿瘤抗原或免疫检查点的抗体的轻链或重链相应CDR的氨基酸序列具有至少80％同一性的氨基酸序列；优选地，所述特异性结合肿瘤抗原或免疫检查点的抗体的轻链含氨基酸序列如SEQ ID NO:6所示的LCDR1、氨基酸序列如SEQ ID NO:7所示的LCDR2、氨基酸序列如SEQ ID NO:8所示的LCDR3；优选地，所述特异性结合肿瘤抗原或免疫检查点的抗体的第二重链含氨基酸序列如SEQ ID NO:3所示的HCDR1、氨基酸序列如SEQ ID NO:4所示的HCDR2、氨基酸序列如SEQ ID NO:5所示的HCDR3。
  8. 一种编码权利要求1-7任一项所述的异源二聚体融合蛋白的核酸，一种含所述核酸的载体或质粒，或者一种表达所述载体或质粒的细胞。
  9. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1-7任一项所述的异源二聚体融合蛋白以及至少一种药学上可接受的赋形剂、稀释剂或载体。
  10. 根据权利要求1-7任一项所述异源二聚体融合蛋白的应用，其特征在于，所述应用包括：(a)制备治疗肿瘤疾病的药物，所述肿瘤疾病包括大肠癌、膜腺癌、肺癌、食管癌、前列腺癌、促结缔组织增生性小圆细胞肿瘤、卵巢癌、胃癌、胰腺癌、肝癌、肾癌、乳腺癌、非小细胞肺癌、黑色素瘤、肺泡横纹肌肉瘤、胚胎性横纹肌肉瘤、尤因肉瘤、肾母细胞瘤、神经母细胞瘤、神经节细胞瘤、髓母细胞瘤、高级别胶质瘤、弥漫性内在脑桥胶质瘤、多层菊形团胚胎性肿瘤中的一种或多种；或(b)制备检测肿瘤抗原或免疫检查点和IL-10受体分子的试剂或试剂盒。
PCT/CN2023/105535 2022-07-08 2023-07-03 一种异源二聚体融合蛋白及其应用 WO2024008039A1 (zh)

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