WO2024078479A1 - 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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WO2024078479A1
WO2024078479A1 PCT/CN2023/123701 CN2023123701W WO2024078479A1 WO 2024078479 A1 WO2024078479 A1 WO 2024078479A1 CN 2023123701 W CN2023123701 W CN 2023123701W WO 2024078479 A1 WO2024078479 A1 WO 2024078479A1
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region
amino acid
heavy chain
seq
acid sequence
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French (fr)
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吴崇兵
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
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a heterodimer fusion protein and an application thereof.
  • Epidermal growth factor receptor also known as EGFR, ErbB-1, and HER1
  • EGFR epidermal growth factor receptor
  • ErbB-1 HER2/c-neu
  • ErbB-3 Her 3
  • Her 4 ErbB-4
  • EGFR epidermal growth factor
  • TGF ⁇ transforming growth factor alpha
  • HB-EGF amphiregulin
  • betacellulin/BTC betacellulin/BTC
  • epigen epithelial cell mitogen protein
  • EGF epidermal growth factor
  • HB-EGF transforming growth factor alpha
  • epigen epithelial cell mitogen protein
  • Y tyrosine residues
  • Mutation, amplification or misregulation of EGFR or family members is involved in approximately 30% of all epithelial cell cancers.
  • mutations that cause EGFR overexpression or hyperreactivity are associated with many cancers, including colon cancer, lung cancer, anal cancer, head and neck cancer, and glioblastoma multiforme.
  • EGFR has been identified as an oncogene, leading to the need to develop anticancer therapeutics for EGFR.
  • Immunomodulators produced by cells of the immune system can directly or indirectly activate cells of the adaptive immune response and can play an important role in eliciting protective anti-tumor immunity.
  • the innate immune system can be triggered by bacterial products or "danger" signals that cause the release of proinflammatory cytokines (such as IFN- ⁇ , TNF- ⁇ , and interleukins).
  • IL-10 is mainly secreted by activated T cells and antigen presenting cells. During antigen recognition, the expression of IL-10 receptor (IL-10R) in CD8+ T cells is upregulated. 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 II Cytokine receptor family (CRF2).
  • IL-10 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 proliferation of CD8+T cells inside the tumor; b. IL-10 can increase the activity and proliferation of antigen-specific T lymphocytes inside the tumor; c. IL-10 has a memory function in its rejection of tumors. Animal in vivo test data show that after the tumor disappears after the IL-10 drug is given, the tumor cells are inoculated into the mice again, and the tumor cells do not grow in the mice. The main reason is that IL-10 can enhance the survival rate of antigen-specific CD8+T cells and play the role of tumor vaccine; d. IL-10 reactivates T cells by restoring the oxidative phosphorylation metabolism of terminal exhausted T cells to achieve the purpose of killing tumor cells.
  • IL-10 can promote the proliferation and survival of CD8+T cells targeting specific antigens, and specific antigen CD8+T cells are positively correlated with immune cells killing tumors.
  • immunomodulators can be used to exert anti-tumor effects in animal models and cancer patients, the short half-life and systemic toxicity associated with the use of immunomodulators have greatly limited their use. After IL-10 binds to the receptor, it activates the STAT3 and STAT1 pathways, which are the signal transduction pathways for IL-10 to exert its biological functions.
  • Cancer is a common disease that endangers human health.
  • the incidence of digestive tract cancer accounts for more than a quarter of common malignant tumors, and the incidence and mortality of digestive tract cancer in my country are the highest.
  • colon cancer has an insidious onset, often without obvious clinical symptoms in the early stage, and the disease progresses slowly. Most patients have already reached the middle and late stages when they show obvious symptoms. At present, no ideal drug for the treatment of digestive tract cancer has been developed.
  • Cetuximab is a monoclonal antibody developed by Merck that targets epidermal growth factor receptors. It was approved for marketing by the U.S. Food and Drug Administration (FDA) on February 12, 2004. Cetuximab can inhibit the ability of tumor cells to repair damage caused by chemotherapy and radiotherapy, as well as inhibit the formation of new blood vessels in tumors. It can be used to treat metastatic colorectal cancer with wild-type RAS and BRAF genes. However, cetuximab has a single target and its effect is not significant enough. Therefore, it is still necessary to find drugs that are significantly better than cetuximab for digestive tract cancer.
  • FDA U.S. Food and Drug Administration
  • the present invention provides a heterodimeric fusion protein and its use.
  • the heterodimeric fusion protein of the present invention can target digestive tract cancer, especially colon cancer, has good targeting, and has a significantly better tumor inhibition effect than cetuximab with the same target.
  • the present invention provides a use of a heterodimeric fusion protein in the preparation of a drug for treating or preventing gastrointestinal cancer
  • the heterodimeric fusion protein comprises: a first heavy chain, wherein the first heavy chain comprises a first Fc region and an immunomodulator fused to the first Fc region; a light chain and a second heavy chain, wherein the second heavy chain comprises a second Fc region, and 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.
  • the tumor antigen or immune checkpoint is B7H3, B7H4, B7H5, Claudin18.2, 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, C D278, CD279, CD357, DR3, Galectin-9, ICOSL/B7RP1/B7H2, IDO, TIGIT, TIM-3, TL1A, gp100, TRP-1, TRP-2, BAGE, GAGE-1, GAGE-2, p15, CEA, Ras, HER-2/neu, MAGE-4, MAGE-5, MAGE-6, RAGE, erbB, p185erbB2, p180erbB-3, c-met, CAM 17.1, NuMa ⁇
  • the light chain and the second heavy chain both comprise a complementary determining region, wherein the complementary determining region comprises 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 comprises LCDR1 as shown in SEQ ID NO:6, LCDR2 as shown in SEQ ID NO:7, and LCDR3 as shown in SEQ ID NO:8; preferably, the second heavy chain comprises amino acids
  • the HCDR1 sequence is shown in SEQ ID NO:3, the HCDR2 amino acid sequence is shown in SEQ ID NO:4, and the HCDR3 amino acid sequence is shown in SEQ ID NO:5.
  • the light chain comprises a variable region
  • 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 has at least 80% identity with SEQ ID NO:10.
  • the second heavy chain comprises a variable region
  • the amino acid sequence of the variable region of the heavy chain is as shown in SEQ ID NO:9, or is an amino acid sequence that is at least 80% identical to SEQ ID NO:9.
  • the amino acid sequence of the light chain is as shown in SEQ ID NO:13, or is an amino acid sequence that has at least 80% identity with SEQ ID NO:13.
  • the amino acid sequence of the second heavy chain is as shown in SEQ ID NO:12, or is an amino acid sequence that has at least 80% identity with SEQ ID NO:12.
  • the first heavy chain comprises one or more immunomodulators of the same or different types.
  • the immunomodulators are fused to each other and to the first Fc region.
  • the immunomodulator is IL-10.
  • the first Fc region is fused to the immunomodulator via a polypeptide linker.
  • the immunomodulator is connected to the N-terminus of the first Fc region via a polypeptide linker.
  • the polypeptide linker is 5-30 amino acids.
  • the first Fc region and the second Fc region are the same or different. In some embodiments, the first Fc region and the second Fc region are selected from IgG, IgA, IgD, IgE, IgM and variants thereof. In some embodiments, the first Fc region and the second Fc region are selected from IgG1, IgG2, IgG3, IgG4 and variants thereof. In some embodiments, the first Fc region and the second Fc region comprise one or more amino acid mutations, preferably amino acid substitutions, insertions or deletions. In some embodiments, the first Fc region is knob-Fc and the second Fc region is hole-Fc.
  • the first Fc region is hole-Fc and the second Fc region is knob-Fc.
  • the amino acid sequence of the first heavy chain is as shown in SEQ ID NO: 14, or is an amino acid sequence having at least 80% identity with SEQ ID NO: 14.
  • the amino acid sequence of the linker monomer in the first heavy chain is as shown in SEQ ID NO: 1.
  • the amino acid sequence of IL-10 in the first heavy chain is as shown in SEQ ID NO: 2.
  • the amino acid sequence of the Fc region in the first heavy chain is as shown in SEQ ID NO: 11.
  • the digestive tract cancer is colon cancer.
  • the present invention also provides the above heterodimer fusion protein.
  • the present invention also provides the above heterodimer fusion protein, which is used for treating or preventing digestive tract cancer.
  • the present invention also provides an isolated polynucleotide encoding the heterodimer fusion protein.
  • the present invention also provides a vector comprising the isolated polynucleotide.
  • the present invention also provides an isolated host cell comprising the isolated polynucleotide or vector.
  • the present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the heterodimer fusion protein.
  • the pharmaceutical composition is formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at a tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous depot.
  • the present invention also provides use of the heterodimeric fusion protein in the preparation of a drug and/or a kit for inhibiting tumors.
  • the tumor is colon cancer.
  • the present invention also provides a method for producing a fusion protein, comprising (i) culturing a host cell under conditions that achieve expression of the fusion protein, and (ii) harvesting the expressed fusion protein.
  • the present invention also provides the use of the heterodimer fusion protein in preparing a reagent or a kit for detecting tumor antigens or immune checkpoints and IL-10 receptor molecules.
  • 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.
  • the two different members may contain 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 contain one, two, or more unit, polypeptide chain or part.
  • fusion protein refers to a protein that includes one, two or more polypeptides derived from different naturally occurring proteins or engineered proteins that are artificially combined to form a protein. Including but not limited to the following forms: 1. For a fusion protein composed of one polypeptide chain, it is composed of the same or different polypeptides fused to each other to form a polypeptide chain containing the different polypeptides; 2.
  • a fusion protein composed of two or more polypeptide chains, wherein one or more optional polypeptide chains are composed of the same or different polypeptides fused to each other to form a polypeptide chain containing the same or different polypeptides, and these polypeptide chains are combined with each other in a covalent or non-covalent form to form a protein.
  • 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 can be an antibody, an antigen-binding antibody fragment, a bispecific antibody or other antibody-based molecules or compounds.
  • 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.
  • 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.
  • 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.
  • tumor antigen may also refer to a biological molecule (e.g., protein, carbohydrate, glycoprotein, etc.) that is uniquely or preferentially or differentially expressed on a cancer cell and/or is found to be associated with a cancer cell, thereby providing a preferential or specific target for cancer.
  • preferential expression may be preferential expression compared to any other cell in an organism, or preferential expression in a specific region of an organism (e.g., in a specific organ or tissue).
  • inhibitory molecules include PDL1, B7H3, CTLA4, etc.
  • activating molecules include OX40, 4-1BB, CD40, etc.
  • an immunomodulator generally refers to a substance that affects the function of the immune system.
  • An immunomodulator can enhance or reduce an immune response.
  • 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/natural preparations of dextran.
  • the immunomodulator is a cytokine.
  • polypeptide linker generally refers to a synthetic amino acid sequence that connects or couples two polypeptide sequences (e.g., couples two polypeptide domains).
  • a polypeptide linker can connect two amino acid sequences via a peptide bond.
  • the polypeptide linker of the present application connects an immunomodulator to an Fc region.
  • antibody generally refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes or immunoglobulin gene fragments.
  • Immunoglobulin genes may include ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ and ⁇ constant region genes, as well as numerous immunoglobulin variable region genes.
  • light chains may be classified as ⁇ or ⁇ .
  • Heavy chains may be classified as ⁇ , ⁇ , ⁇ , ⁇ or ⁇ , which in turn define immunoglobulin classes: IgG, IgM, IgA, IgD and IgE, respectively.
  • the antibodies used in this application may have a structural unit comprising a tetramer.
  • Each tetramer may be composed of two pairs of identical polypeptide chains, each pair having a "light” chain (about 25 kD) and a "heavy chain” (about 50-70 kD).
  • the N-terminus of each member may define a variable region of about 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 light and heavy chains, respectively.
  • Antibodies may exist as complete immunoglobulins or as many well-characterized fragments produced 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 a recombinant DNA method, including but not limited to Fab'2, IgG, IgM, IgA, IgE, scFv, dAb, nanobodies, single antibodies and double-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 the variable heavy chain and the variable light chain (directly or through a peptide linker) are connected together to form a continuous polypeptide.
  • the antibodies and fragments in the present application are bispecific.
  • bispecific antibodies or fragments thereof have binding specificity to at least two different epitopes (e.g., at least one of 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 include two or more antibodies or antibody binding fragments (e.g., Fab) connected together, wherein each antibody or fragment has a different specificity.
  • CDRs There are multiple 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, defining 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 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 (eg, amino acid residues in the Fc region) and target regions (eg, CDRs) in the present invention uses the Kabat system.
  • identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percentage sequence identity. Comparisons for purposes of determining percentage 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 FASTA packages.
  • 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 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%.
  • the term "host cell” generally includes a single cell, cell line or cell culture that may be or has been a recipient of a subject's plasmid or vector, which contains polynucleotides disclosed in the present application, or expresses a heterodimer fusion protein of the present application.
  • a host cell may include the offspring of a single host cell. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical (morphologically or on the genomic total DNA complement) to the original parent cell.
  • a host cell may include a cell transfected in vitro with a vector disclosed in the present application.
  • a host cell may be a bacterial cell (e.g., Escherichia coli (E. coli)), a yeast cell or other eukaryotic cells, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell or a myeloma cell.
  • vector generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into a host cell and/or between host cells.
  • the term may include vectors primarily used to insert DNA or RNA into cells, vectors primarily used for replication of DNA or RNA, and expression vectors for 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 an approach to obtaining beneficial or desired results, including but not limited to therapeutic benefit and/or preventive benefit.
  • a therapeutic benefit generally refers to eradication or lessening of the severity of the underlying condition being treated.
  • a therapeutic benefit is achieved by eradication, lessening of severity, or reduction in 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).
  • a preventive benefit a subject at risk of developing a particular disease, or reporting a disease, may be given a course of treatment that is appropriate for that subject.
  • the compositions may be administered to a subject who is experiencing one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.
  • the term "digestive tract cancer” refers to a tumorous lesion that occurs in the digestive system. Most of them need to be named according to the location of the tumor, benign or malignant, etc., such as esophageal cancer, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, small intestine cancer, large intestine cancer, gallbladder cancer, pancreatic cancer, etc.
  • EGFR epidermal growth factor receptor
  • the present invention preferably overexpresses EGFR protein in digestive tract cancer, especially overexpresses EGFR protein in colon cancer.
  • FIG1 is a fitting curve of the binding activity of the fusion protein to the human EGFR protein.
  • FIG. 2 is a fitting curve of the binding activity of the fusion protein to IL-10Ra.
  • FIG. 3 is a curve showing the affinity of the fusion protein to the EGFR protein.
  • FIG. 4 is a curve showing the affinity of the fusion protein to the IL-10R protein.
  • FIG5 is a peak diagram of the dual-target binding activity detection of the fusion protein and MDA-MB-231 cells.
  • FIG6 is a peak diagram of the dual-target binding activity of the fusion protein and IL-10-Reporter-HEK-293 cells.
  • FIG. 7 is a graph showing the test results of fusion protein activating CD8+ T cells to secrete perforin.
  • FIG8 is a tumor growth curve of MC38-hEGFR transplanted tumor model bearing mice after administration of the test fusion protein.
  • the target amino acid sequence was converted into nucleotide sequence, and the obtained nucleotide sequences were: SEQ ID NO: 15 (first heavy chain), SEQ ID NO: 16 (light chain), and SEQ ID NO: 17 (second heavy chain).
  • the pcDNA3.4-G418 and pcDNA3.1-G418 vectors are used as special vectors for expressing the light chain and heavy chain of the multifunctional antibody, respectively.
  • the pcDNA3.4-G418 vector contains the promoter CMVPromoter, the eukaryotic screening marker G418 tag and the prokaryotic screening marker Ampicilline used for the light chain.
  • the pcDNA3.1-G418 vector contains the promoter CMVPromoter, the eukaryotic screening marker G418 tag and the prokaryotic screening marker Ampicilline used for the heavy chain. Prokaryotic screening tag Ampicilline.
  • the nucleotide sequences of the antibody expression light chain and heavy chain were obtained by gene synthesis, and the vector and the target fragment were double-digested with HindIII and XhoI, and then enzymatically linked with DNA ligase after recovery, and transformed into Escherichia coli competent cells DH5 ⁇ , and positive clones were selected and plasmids were extracted and enzyme-digested to obtain recombinant plasmids containing the first heavy chain, the second heavy chain, and the light chain of the antibody, which were pcDNA3.1-G418-1, pcDNA3.1-G418-2, and pcDNA3.4-G418-3, respectively.
  • the recombinant plasmids containing the above-mentioned target genes were transformed into Escherichia coli competent cells DH5 ⁇ , the transformed bacteria were spread on LB plates containing 100 ⁇ g/mL ampicillin and cultured, the plasmid clones were selected and cultured in liquid LB culture medium, and the bacteria were shaken at 260 rpm for 14 hours.
  • 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.
  • Example 4 Plasmid transfection, transient expression and fusion protein purification
  • ExpiCHO was cultured at 37°C, 8% CO 2 , 100 rpm to a cell density of 6 ⁇ 10 6 cells/mL.
  • the constructed vectors PCDNA3.1-G418-6-1, PCDNA3.1-G418-6-2, and PCDNA3.4-G418-6-3 were transfected into the above cells using liposomes, respectively.
  • the transfection plasmid concentration was 1 ⁇ g/mL, and the liposome concentration was determined with reference to the ExpiCHO TM Expression System kit.
  • the cells were cultured at 32°C, 5% CO 2 , and 100 rpm for 7-10 days. Feed was performed once 18-22 hours after transfection and between 5-8 days.
  • the culture product was centrifuged at 4000 rpm, filtered through a 0.22 ⁇ m filter membrane, and the culture supernatant was collected.
  • the antibody protein obtained was purified using Protein A and an ion column, and the eluate was collected.
  • 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 equilibrium buffer used for chromatography is 1 ⁇ PBS (pH7.4). After the cell supernatant is loaded and combined, it is washed with PBS until the ultraviolet returns to the baseline, and then the target protein is eluted with an elution buffer of 0.1M glycine (pH3.0), and the pH is adjusted to neutral with Tris for storage.
  • the pH of the product obtained by affinity chromatography is adjusted to 1-2 pH units lower or higher than the pI, and appropriately diluted to control the sample conductivity below 5ms/cm.
  • 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 target protein is selected according to SDS-PAGE and SEC-HPLC.
  • the collection tubes containing the proteins were combined and saved.
  • Example 5 Binding ability of fusion protein to EGFR and IL-10 receptor protein
  • the ELISA method was used to detect the binding ability of the fusion protein to human EGFR and IL-10R proteins, and the SPR technology was used to detect the affinity of the fusion protein to EGFR and IL-10R proteins.
  • the ELISA test results showed that the EGFR binding end of the fusion protein can specifically bind to human EGFR, and the IL-10R binding end of the fusion protein can specifically bind to human IL-10Ra.
  • the binding activity data are shown in Table 1, Figures 1 and 2. As shown in Figures 3 and 4, the affinity of the fusion protein to EGFR and IL-10Ra proteins is 0.51nM and 3.38nM, respectively.
  • the positive control is cetuximab
  • the heavy chain and light chain amino acid sequences of negative control 1 are shown in SEQ ID NO: 18 and SEQ ID NO: 19, respectively
  • negative control 2 is the commercially available Human IgG1kappa Isotype Control of Sino, HGK1.
  • This experiment uses the specific binding properties between antigen and antibody and ligand and receptor, uses anti-HIS-tagged FITC fluorescent antibody, and uses FACS technology to analyze the simultaneous binding of fusion protein to EGFR and IL-10R.
  • MDA-MB-231 cells are natural tumor cells that express EGFR antigen, and it has been verified that they bind to the EGFR end of the fusion protein.
  • IL-10Reporter-HEK-293 tool cells stably express IL-10R and STAT3 signaling pathway reporter gene system, which can bind to the IL-10 end of the fusion protein.
  • FIG. 5 and 6 they are peak graphs of the dual-target binding activity detection of the fusion protein and MDA-MB-231 cells and peak graphs of the dual-target binding activity of the fusion protein and IL-10-Reporter-HEK-293 cells, respectively.
  • the experiment set up blank control group (Blank), negative control group (NC), experimental group (fusion protein
  • the experimental group, single target control group (cetuximab, Human-IL-10) and isotype control group (Human-IgG1) were first mixed with Human-IL-10RA Protein (His Tag) protein, and then incubated with MDA-MB-231 cells.
  • Anti-6X-His tag antibody (FITC) was used as the secondary antibody for incubation for 0.5h, and the secondary antibody binding signal was detected by flow cytometry.
  • the blank control group only added the secondary antibody, and the negative control group was protein diluent.
  • the experiment set up a blank control group (blank), a negative control group (NC), an experimental group (fusion protein), a single target control group (cetuximab, Human-IL-10) and an isotype control group (Human-IgG1); the experimental group, the single target control group, the isotype control group and the negative control group were first mixed with Human-EGFR Protein, His Tag protein, and then incubated with IL-10-Reporter-HEK-293 cells for 1 hour, and then Anti-6X-His tag antibody (FITC) secondary antibody was added, and the secondary antibody binding signal was detected by flow cytometry.
  • the blank control group only added the secondary antibody, and the negative control group was the protein diluent.
  • Human-IgG1 comes from the commercially available Human IgG1kappa Isotype Control of Sino, HGK1.
  • Example 7 CD8 + T cell activation assay using fusion protein
  • the fusion protein is that after its IL-10 end binds to IL-10R in CD8 + T, it activates CD8 + T for tumor cell killing.
  • the fusion protein is reacted with isolated peripheral blood, and then the commercial Perforin cytokine detection kit is used to detect the fusion protein's stimulation of CD8 + T cell secretion of Perforin.
  • IL-10 significantly stimulates CD8 + T cells to secrete perforin, and it is concentration-dependent.
  • the fusion protein antibody can significantly stimulate CD8 + T cells to secrete perforin at high concentrations, while cetuximab cannot stimulate CD8 + T cells to secrete perforin, indicating that the fusion protein stimulates CD8 + T cells to secrete perforin depending on the IL-10 end.
  • One of the mechanisms of action of the fusion protein is that the IL-10 end binds to the IL-10 receptor on the surface of DC cells in the tumor microenvironment, inhibiting the apoptosis of CD8 + T cells induced by DC cells.
  • the in vivo efficacy of the fusion protein was tested in C57BL/6 mice and the tumor cells were mouse colon cancer cells MC38 that overexpressed hEGFR. Cetuximab was used as a positive control.
  • the test fusion protein had significant tumor inhibition effects at doses of 1 mg/kg, 5 mg/kg and 10 mg/kg, with TGIs of 63.05%, 83.26% and 82.46%, respectively, while the TGI of the control group cetuximab was 20.15%, with no significant tumor inhibition effect.
  • the tumor inhibition effect of the fusion protein 10mg/kg group was similar to that of the fusion protein 10mg/kg group, and one animal achieved complete remission of the tumor. Therefore, the tumor inhibition effect of the fusion protein 5mg/kg group was significantly better than that of cetuximab, and the main mechanism of action may be the activation of antigen-specific CD8 + T cells in the tumor microenvironment by IL-10.

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Abstract

本发明涉及一种异源二聚体融合蛋白及其应用。本发明的异源二聚体融合蛋白包含：第一重链，所述第一重链包含第一Fc区及与第一Fc区融合的免疫调节剂；轻链和第二重链，所述第二重链包含第二Fc区，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。本发明的异源二聚体融合蛋白能够靶向治疗消化道癌尤其是结肠癌，靶向性好，抑瘤作用显著优于同靶点的西妥昔单抗。

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
表皮生长因子受体(也称作EGFR、ErbB-1和HER1)是ErbB受体家族的一种细胞表面受体，ErbB受体家族是由四种密切相关的受体酪氨酸激酶组成的亚家族，包括EGFR(ErbB-1)、HER2/c-neu(ErbB-2)、Her 3(ErbB-3)和Her 4(ErbB-4)。EGFR与配体(如表皮生长因子(EGF)、转化生长因子a(TGFα)、HB-EGF、双调蛋白、β细胞素/BTC、上皮细胞有丝分裂蛋白(epigen)/EPGN等)的结合在EGFR的C末端结构域中诱导一些酪氨酸(Y)残基(Y992、Y1045、Y1068、Y1148和Y1173)的受体二聚体化及自身磷酸化。这种自身磷酸化引发包括MAPK、Akt和JNK途径在内的一些信号转导级联的下游激活，导致细胞迁移、粘附和细胞增殖。
EGFR或家族成员的突变、扩增或误调节涉及全部上皮细胞癌的大约30％。例如，导致EGFR过表达或过度反应性的突变与许多癌症相关，包括结肠癌、肺癌、肛门癌、头颈癌及多形性成胶质细胞瘤。EGFR被鉴别为癌基因导致需要开发针对EGFR的抗癌治疗剂。
引起疾病的恶性细胞通常不能引起导致排斥的免疫应答。研究已显示，有可能通过将免疫调节分子(诸如细胞因子和共刺激分子)引入肿瘤细胞来增强其免疫原性；然而，根除残余癌细胞可能需要靶向广泛分散的微转移肿瘤沉积物，而这些微转移肿瘤沉积物不能直接进行基因转移。另外，引入的免疫调节分子的表达和稳定性通常远不令人满意。由免疫系统的细胞产生的免疫调节剂，诸如细胞因子，可以直接或间接激活适应性免疫反应的细胞，并且可在引发保护性抗肿瘤免疫中起重要作用。先天免疫系统可以通过导致促炎症细胞因子(诸如IFN‐α、TNF‐α和白细胞介素)释放的细菌产物或“危险”信号触发。
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，IL-10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.IL-10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.IL-10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。
癌症是危害人体健康的常见多发病，其中消化道癌的发病率占常见恶性肿瘤的四分之一以上，而我国消化道癌的发病率与死亡率最高。其中的结肠癌起病隐匿，早期常无明显的临床症状，而且病情发展较慢，病人出现明显症状时大多已经到了中晚期。目前还没有研发出一种理想的治疗消化道癌症的药物。
西妥昔单抗(Cetuximab)是由默克公司研发的一种靶向作用于表皮生长因子受体的单克隆抗体，于2004年2月12日获美国食品和药物管理局(FDA)批准上市。西妥昔单抗能够抑制肿瘤细胞修复化疗和放疗所致损伤的能力、以及抑制肿瘤内新生血管的形成，可用于治疗RAS、BRAF基因野生型的转移性结直肠癌。但是西妥昔单抗靶点单一，效果不够显著，因此仍然需要寻找针对消化道癌症的效果显著优于西妥昔单抗的药物。
发明内容
为了解决上述技术问题，本发明提供了一种异源二聚体融合蛋白及其用途。本发明的异源二聚体融合蛋白能够靶向治疗消化道癌尤其是结肠癌，靶向性好，抑瘤作用显著优于同靶点的西妥昔单抗。
具体而言，本发明提供一种异源二聚体融合蛋白在制备用于治疗或预防消化道癌的药物中的用途，所述异源二聚体融合蛋白包含：第一重链，所述第一重链包含第一Fc区及与所述第一Fc区融合的免疫调节剂；轻链和第二重链，所述第二重链包含第二Fc区，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。
在一些实施方案中，所述肿瘤抗原或免疫检查点为B7H3、B7H4、B7H5、Claudin18.2、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、ICOSL/B7RP1/B7H2、IDO、TIGIT、TIM-3、TL1A、gp100、TRP-1、TRP-2、BAGE、GAGE-1、GAGE-2、p15、CEA、Ras、HER-2/neu、MAGE-4、MAGE-5、MAGE-6、RAGE、erbB、p185erbB2、p180erbB-3、c-met、CAM 17.1、NuMa、K-ras、CDK4、Mum-1、p 15、p 16、43-9F、5T4、791Tgp72、β-HCG、BCA225、BTAA、CA 125、MUC16、IL13Rα2、FRα、VEGFR2、LewisY、FAP、EphA2、CEACAM5、CEACAM6、EGFR、CA6、CA9、GPNMB、EGP1、FOLR1、STEAP1、SLC44A4、AGS-16、MUC-1、CFC1B、TPS、CD19、CD20、CD22、CD30、CD72、CD180、CD171、CD123、CD133、CD138、CD37、CD70、CD79a、CD79b、CD56、CD74、CD166、CD71、CLL-1/CLEC12A、ROR1、CD33/IL3Ra、c-Met、PSCA、PSMA、EGFRvIII、BCMA、GD-2、MY-ESO-1或MAGE A3中的一种或多种；优选地，所述肿瘤抗原为EGFR。
在一些实施方案中，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，所述轻链包含氨基酸序列如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，所述第二重链包含氨基酸 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。
在一些实施方案中，所述轻链包含可变区，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。
在一些实施方案中，所述第二重链包含可变区，所述重链的可变区的氨基酸序列如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。
在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，所述第一重链包含一个或多个相同或不同类型的免疫调节剂。在一些实施方案中，所述免疫调节剂相互融合且与所述第一Fc区融合。在一些实施方案中，所述免疫调节剂是IL-10。
在一些实施方案中，所述第一Fc区通过多肽接头与所述免疫调节剂融合。在一些实施方案中，所述免疫调节剂通过多肽接头连接至所述第一Fc区的N端。在一些实施方案中，所述多肽接头为5-30个氨基酸。在一些实施方案中，所述多肽接头为(GGGGS)n，其中n＝1-6。
在一些实施方案中，所述第一Fc区和所述第二Fc区是相同或不同的。在一些实施方案中，所述第一Fc区和所述第二Fc区选自IgG、IgA、IgD、IgE、IgM及其变体。在一些实施方案中，所述第一Fc区和所述第二Fc区选自IgG1、IgG2、IgG3、IgG4及其变体。在一些实施方案中，所述第一Fc区和所述第二Fc区包含一个或多个氨基酸突变，优选氨基酸置换、插入或缺失。在一些实施方案中，所述第一Fc区为knob-Fc，所述第二Fc区为hole-Fc。在一些实施方案中，所述第一Fc区为hole-Fc，所述第二Fc区为knob-Fc。在一些实施方案中，所述第一重链的氨基酸序列如SEQ ID NO:14所示，或为与SEQ ID NO:14具有至少80％同一性的氨基酸序列。
在一些实施方案中，第一重链中的接头单体的氨基酸序列如SEQ ID NO:1所 示。在一些实施方案中，第一重链中的IL-10的氨基酸序列如SEQ ID NO:2所示。在一些实施方案中，第一重链中的Fc区的氨基酸序列如SEQ ID NO:11所示。
在一些实施方案中，所述消化道癌为结肠癌。
本发明还提供上述异源二聚体融合蛋白。
本发明还提供上述异源二聚体融合蛋白，其用于治疗或预防消化道癌。
本发明还提供一种分离的多核苷酸，其编码上述异源二聚体融合蛋白。
本发明还提供一种载体，其包含分离的多核苷酸。
本发明还提供一种分离的宿主细胞，其包含分离的多核苷酸或载体。
本发明还提供一种药物组合物，其包含药学上可接受的赋形剂和上述异源二聚体融合蛋白。
在一些实施方案中，所述药物组合物被配制用于口服施用，静脉内施用，肌内施用，在肿瘤部位的原位施用，吸入，直肠施用，阴道施用，经皮施用或通过皮下储库施用。
本发明还提供上述异源二聚体融合蛋白在制备用于抑制肿瘤药剂和/或试剂盒中的用途，优选地，所述肿瘤为结肠癌。
本发明还提供一种产生融合蛋白的方法，其包括(i)在实现所述融合蛋白表达的条件下培养宿主细胞，和(ii)收获表达的融合蛋白。
本发明还提供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中的应用。
术语的详细说明
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
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“融合蛋白”是指包括一种、两种或更多种源自不同天然存在的蛋白质或者工程化改造后的蛋白质的多肽被人为地组合以形成一种蛋白质的蛋白质。包括但不限于一下例举的形式，1、对于一条多肽链组成的融合蛋白，其由相同或不同的多肽彼此融合，以形成包含所述不同的多肽的一条多肽链；2、对于两条或以上多肽链组成的融合蛋白，其中任选的一条或多条多肽链由相同或不同的多肽彼此融合，以形成包含所述相同或不同的多肽的多肽链，这些多肽链以共价或非共价的形式彼此组合形成蛋白。
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“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。
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“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融合蛋白。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“消化道癌”是指发生于消化系统中的肿瘤性病变。大多数需要根据肿瘤发生的部位、良恶性等来命名，例如食管癌，胃癌，结肠癌，直肠癌，胰腺癌，胆囊癌，胆管癌、小肠癌、大肠癌、胆嚢癌、胰腺癌等。在消化道癌中，表皮生长因子受体(EGFR)基因扩增及其蛋白过表达十分常见，且蛋白过表达还具有预后指导意义。在一些实施方案中，本发明优选EGFR蛋白过表达的消化道癌，尤其是EGFR蛋白过表达的结肠癌。
附图说明
图1是融合蛋白与人EGFR蛋白的结合活性拟合曲线。
图2是融合蛋白与IL-10Ra的结合活性拟合曲线。
图3是融合蛋白与EGFR蛋白亲和力结果曲线。
图4是融合蛋白与IL-10R蛋白亲和力结果曲线。
图5是融合蛋白与MDA-MB-231细胞双靶点结合活性检测峰图。
图6是融合蛋白与IL-10-Reporter-HEK-293细胞双靶点结合活性峰图。
图7是融合蛋白激活CD8+T细胞分泌穿孔蛋白试验结果图。
图8是MC38-hEGFR移植瘤模型荷瘤鼠在给予受试物融合蛋白的肿瘤生长曲线。
具体实施方式
实施例1：核苷酸序列
将目标氨基酸序列转化为核苷酸序列，获得核苷酸序列分别为：SEQ ID NO:15(第一重链)、SEQ ID NO:16(轻链)、SEQ ID NO:17(第二重链)。
实施例2：基因合成与表达载体的构建
分别采用pcDNA3.4-G418和pcDNA3.1-G418载体作为表达所述多功能抗体的轻链和重链的专用载体。pcDNA3.4-G418含有轻链所使用的启动子CMVPromoter、真核筛选标记G418标签和原核筛选标签Ampicilline。pcDNA3.1-G418载体含有重链所使用的启动子CMVPromoter、真核筛选标记G418标签和 原核筛选标签Ampicilline。基因合成得到抗体表达轻链和重链的核苷酸序列，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，获得含所述抗体第一重链、第二重链、轻链重组质粒，分别为pcDNA3.1-G418-1、pcDNA3.1-G418-2和pcDNA3.4-G418-3。
实施例3：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14小时，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4：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个细胞/mL。使用脂质体分别将构建的载体PCDNA3.1-G418-6-1、PCDNA3.1-G418-6-2、PCDNA3.4-G418-6-3转染到上述细胞中，转染质粒浓度为1μg/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5-8天之间分别补料一次。4000rpm离心上述培养产物，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(pH7.4)，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(pH3.0)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pI 1-2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及SEC-HPLC选择目的 蛋白所在的收集管合并保存。
实施例5：融合蛋白与EGFR和IL-10受体蛋白结合能力
采用ELISA的方法，检测融合蛋白与人EGFR和IL-10R蛋白的结合能力，并利用SPR技术检测融合蛋白分别与EGFR和IL-10R蛋白的亲和力。ELISA检测结果显示，融合蛋白的EGFR结合端可以特异性结合人EGFR，融合蛋白的IL-10R结合端可以特异性结合人IL-10Ra。结合活性数据见表1，图1和图2。如图3和4所示，融合蛋白与EGFR、IL-10Ra蛋白的亲和力分别为0.51nM和3.38nM。其中阳性对照为西妥昔单抗，阴性对照1的重链和轻链氨基酸序列分别如SEQ ID NO:18和SEQ ID NO:19所示，阴性对照2为市售Sino,HGK1的Human IgG1kappa Isotype Control。
表1融合蛋白与人EGFR蛋白和人IL-10R的体外结合活性
实施例6：双靶点结合试验
本试验利用抗原与抗体及配体与受体之间的特异性结合特性，使用抗HIS标签的FITC荧光抗体，采用FACS技术分析融合蛋白与EGFR和IL-10R的同时结合。MDA-MB-231细胞是表达EGFR抗原的天然肿瘤细胞，已验证其与融合蛋白的EGFR端有结合。IL-10Reporter-HEK-293工具细胞稳定表达IL-10R及STAT3信号通路报告基因系统，可与融合蛋白的IL-10端结合。研究结果显示，融合蛋白IL-10端与IL-10RA蛋白结合后，仍然与EGFR阳性MDA-MB-231细胞的EGFR结合，而融合蛋白与EGFR蛋白结合后，仍然与IL-10-Reporter-HEK-293细胞结合。该试验证明了融合蛋白与其两端作用靶点可同时结合，融合蛋白通过其抗EGFR抗体将IL-10导入到其肿瘤微环境后，IL-10可以有效的和作用细胞结合，发挥其生物活性作用。如图5和图6所示，分别为融合蛋白与MDA-MB-231细胞双靶点结合活性检测峰图和融合蛋白与IL-10-Reporter-HEK-293细胞双靶点结合活性峰图。
图5中，实验设置空白对照组(Blank)、阴性对照组(NC)、实验组(融合蛋 白)、单靶点对照组(西妥昔单抗、Human-IL-10)和同型对照组(Human-IgG1)；实验组、单靶点对照组、同型对照组、阴性对照组首先与Human-IL-10RA Protein(His Tag)蛋白混合，然后再与MDA-MB-231细胞孵育，采用Anti-6X-His tag antibody(FITC)二抗孵育0.5h，流式细胞仪检测二抗结合信号。空白对照组只加二抗，阴性对照组为蛋白稀释液。图6中，实验设置空白对照组(blank)、阴性对照组(NC)、实验组(融合蛋白)、单靶点对照组(西妥昔单抗、Human-IL-10)和同型对照组(Human-IgG1)；实验组、单靶点对照组、同型对照组和阴性对照组首先与Human-EGFR Protein,His Tag蛋白混合，然后与IL-10-Reporter-HEK-293细胞孵育1h后，加入Anti-6X-His tag antibody(FITC)二抗，流式细胞仪检测二抗结合信号。空白对照组只加二抗，阴性对照组为蛋白稀释液。Human-IgG1来源于市售Sino,HGK1的Human IgG1kappa Isotype Control。
实施例7：融合蛋白的CD8+T细胞激活试验
融合蛋白的主要作用机制之一为其IL-10端与CD8+T中IL-10R结合后，激活CD8+T用于肿瘤细胞的杀伤。首先将融合蛋白与分离的外周血作用，然后利用商业Perforin细胞因子检测试剂盒检测融合蛋白对CD8+T细胞的刺激Perforin分泌情况。如图7所示，IL-10较显著的刺激CD8+T细胞分泌穿孔蛋白，且具有浓度依赖性，融合蛋白抗体在高浓度可以明显的刺激CD8+T细胞分泌穿孔蛋白，而西妥昔单抗不能刺激CD8+T细胞分泌穿孔蛋白，提示融合蛋白刺激CD8+T细胞分泌穿孔蛋白作用依赖于IL-10端。
实施例8：融合蛋白体内药效学研究
基于融合蛋白的作用机制之一为IL-10端与肿瘤微环境DC细胞表面IL-10受体结合，抑制DC细胞所诱导的CD8+T细胞的凋亡，融合蛋白的体内药效采用的小鼠为C57BL/6，肿瘤细胞为过表达hEGFR的小鼠结肠癌细胞MC38。以西妥昔单抗作为阳性对照。
如图8所示，相比较于Saline对照组，受试物融合蛋白在1mg/kg、5mg/kg和10mg/kg剂量下均具有显著的抑瘤作用，TGI分别为63.05％、83.26％和82.46％,而对照组西妥昔单抗的TGI为20.15％，无明显的抑瘤作用。融合蛋白5mg/kg组 的抑瘤作用与融合蛋白10mg/kg组相近，均有1只动物肿瘤达到完全缓解。因此，融合蛋白5mg/kg组的抑瘤作用显著优于西妥昔单抗，主要的作用机制可能为IL-10对肿瘤微环境中抗原特异性CD8+T细胞的激活作用。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。

Claims (24)

  1. 一种异源二聚体融合蛋白在制备用于治疗或预防消化道癌的药物中的用途，其特征在于，所述异源二聚体融合蛋白包含：第一重链，所述第一重链包含第一Fc区及与所述第一Fc区融合的免疫调节剂；轻链和第二重链，所述第二重链包含第二Fc区，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。
  2. 根据权利要求1所述的用途，所述肿瘤抗原或免疫检查点为B7H3、B7H4、B7H5、Claudin18.2、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、ICOSL/B7RP1/B7H2、IDO、TIGIT、TIM-3、TL1A、gp100、TRP-1、TRP-2、BAGE、GAGE-1、GAGE-2、p15、CEA、Ras、HER-2/neu、MAGE-4、MAGE-5、MAGE-6、RAGE、erbB、p185erbB2、p180erbB-3、c-met、CAM 17.1、NuMa、K-ras、CDK4、Mum-1、p 15、p 16、43-9F、5T4、791Tgp72、β-HCG、BCA225、BTAA、CA 125、MUC16、IL13Rα2、FRα、VEGFR2、LewisY、FAP、EphA2、CEACAM5、CEACAM6、EGFR、CA6、CA9、GPNMB、EGP1、FOLR1、STEAP1、SLC44A4、AGS-16、MUC-1、CFC1B、TPS、CD19、CD20、CD22、CD30、CD72、CD180、CD171、CD123、CD133、CD138、CD37、CD70、CD79a、CD79b、CD56、CD74、CD166、CD71、CLL-1/CLEC12A、ROR1、CD33/IL3Ra、c-Met、PSCA、PSMA、EGFRvIII、BCMA、GD-2、MY-ESO-1或MAGE A3中的一种或多种；优选地，所述肿瘤抗原为EGFR。
  3. 根据权利要求2所述的用途，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。
  4. 根据权利要求1所述的用途，其特征在于，所述轻链包含可变区，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。
  5. 根据权利要求1所述的用途，其特征在于，所述第二重链包含可变区，所述重链的可变区的氨基酸序列如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。
  6. 根据权利要求1所述的用途，其特征在于，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。
  7. 根据权利要求1所述的用途，其特征在于，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。
  8. 根据权利要求1所述的用途，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第一Fc区融合。
  9. 根据权利要求8所述的用途，其特征在于，所述免疫调节剂是IL-10。
  10. 根据权利要求8或9所述的用途，其特征在于，所述第一Fc区和所述第二Fc区是相同或不同的。
  11. 根据权利要求10所述的用途，其特征在于，所述第一Fc区和所述第二Fc区选自IgG、IgA、IgD、IgE、IgM及其变体。
  12. 根据权利要求11所述的用途，其特征在于，所述第一Fc区和所述第二Fc区选自IgG1、IgG2、IgG3、IgG4及其变体。
  13. 根据权利要求11或12所述的用途，其特征在于，所述第一Fc区和所述第二Fc区包含一个或多个氨基酸突变，优选氨基酸置换、插入或缺失。
  14. 根据权利要求10-13任一项所述的用途，其特征在于，所述第一Fc区为knob-Fc，所述第二Fc区为hole-Fc。
  15. 根据权利要求10-13任一项所述的用途，其特征在于，所述第一Fc区为hole-Fc，所述第二Fc区为knob-Fc。
  16. 根据权利要求1所述的用途，其特征在于，所述第一重链的氨基酸序列如SEQ ID NO:14所示，或为与SEQ ID NO:14具有至少80％同一性的氨基酸序列。
  17. 根据权利要求1所述的用途，其特征在于，所述消化道癌为结肠癌。
  18. 如权利要求1-17任一项所述的异源二聚体融合蛋白。
  19. 如权利要求18所述的异源二聚体融合蛋白，其用于治疗或预防消化道癌。
  20. 一种分离的多核苷酸，其编码权利要求18或19的异源二聚体融合蛋白。
  21. 一种载体，其包含权利要求20的分离的多核苷酸。
  22. 一种分离的宿主细胞，其包含权利要求20的分离的多核苷酸或权利要求21的载体。
  23. 一种药物组合物，其包含药学上可接受的赋形剂和权利要求18或19的异源二聚体融合蛋白。
  24. 根据权利要求23所述的药物组合物，其被配制用于口服施用，静脉内施用，肌内施用，在肿瘤部位的原位施用，吸入，直肠施用，阴道施用，经皮施用或通过皮下储库施用。
PCT/CN2023/123701 2022-10-10 2023-10-10 一种异源二聚体融合蛋白及其应用 WO2024078479A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111132998A (zh) * 2017-09-25 2020-05-08 苏州丁孚靶点生物技术有限公司 用于癌症治疗的方法和组合物
WO2022140797A1 (en) * 2020-12-23 2022-06-30 Immunowake Inc. Immunocytokines and uses thereof
WO2023046047A1 (zh) * 2021-09-27 2023-03-30 盛禾(中国)生物制药有限公司 一种异源二聚体蛋白质及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111132998A (zh) * 2017-09-25 2020-05-08 苏州丁孚靶点生物技术有限公司 用于癌症治疗的方法和组合物
WO2022140797A1 (en) * 2020-12-23 2022-06-30 Immunowake Inc. Immunocytokines and uses thereof
WO2023046047A1 (zh) * 2021-09-27 2023-03-30 盛禾(中国)生物制药有限公司 一种异源二聚体蛋白质及其应用

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