[go: up one dir, main page]

WO2019096137A1 - Hybridome comprenant un inhibiteur de point de contrôle immunitaire, procédé de préparation de celui-ci, et applications associées - Google Patents

Hybridome comprenant un inhibiteur de point de contrôle immunitaire, procédé de préparation de celui-ci, et applications associées Download PDF

Info

Publication number
WO2019096137A1
WO2019096137A1 PCT/CN2018/115286 CN2018115286W WO2019096137A1 WO 2019096137 A1 WO2019096137 A1 WO 2019096137A1 CN 2018115286 W CN2018115286 W CN 2018115286W WO 2019096137 A1 WO2019096137 A1 WO 2019096137A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cancer
amino acid
nos
inhibitor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2018/115286
Other languages
English (en)
Chinese (zh)
Inventor
韩化敏
张叔人
高荣凯
褚丽新
徐义
金瑾
魏晓莉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biocells Beijing Biotech Co Ltd
Original Assignee
Biocells Beijing Biotech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biocells Beijing Biotech Co Ltd filed Critical Biocells Beijing Biotech Co Ltd
Publication of WO2019096137A1 publication Critical patent/WO2019096137A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • 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
    • 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
    • A61K38/21—Interferons [IFN]
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • 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
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • 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
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • 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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present application belongs to the field of immunotherapy, and relates to an immunotherapeutic drug for cancer, and particularly relates to a fusion structure of an immunological checkpoint and a cytokine for immunotherapy.
  • Cytokines are important immunotherapeutic products in the field of cancer immunotherapy. Cytokines act as molecular messengers, allowing cells of the immune system to communicate with each other to produce coordination of target antigens to exert regulatory and effector functions in many diseases. As an immunomodulator, cytokines can be used to activate immunotherapy, immunosuppressive therapy, and the like. Clinical application of cytokines for the treatment of cancer and other diseases has been more than 20 years. Cytokines such as interferon (IFN) and interleukin (IL) have been widely used in the treatment of hairy cell leukemia.
  • IFN interferon
  • IL interleukin
  • cytokines mainly act through immune regulation, and overdosing may inhibit the expected immune response, while the dose is too low. It can not effectively cause an immune response; (2) long-term daily medication can induce immune tolerance; (3) the effect shows slow but long-lasting: biological immunotherapy is different from radiotherapy and chemotherapy, even tumors that are sensitive to cytokines, It disappears completely after treatment, and the treatment effect may be displayed after a few months. The condition will continue to improve after the cytokine is stopped.
  • Combination therapy is better than monotherapy: various cytokine combinations Application is more effective than treatment with only one cytokine, because multiple cytokines can make up for each other's shortcomings, and exert their own advantages, so that the effect is better; (5) can prolong the life of patients: cytokines can inhibit tumor cell growth And the toxic side effects are small, so the life of the patient can be significantly prolonged.
  • cytokines are highly efficient, they still have the disadvantages of single component, targeting and killing.
  • the object of the present application is to provide a novel disease treatment product which can combine the high anti-tumor effect of anti-tumor cytokines and the characteristics of immunological checkpoint inhibition to provide a new choice for tumor treatment.
  • embodiments of the present application that achieve the above-described objectives include the following:
  • WHAT IS CLAIMED IS 1. A fusion for immunotherapy comprising an immunological checkpoint inhibitor and a cytokine.
  • the immunological checkpoint inhibitor comprises, but is not limited to, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a guanamine 2,3-double Oxygenase (IDO-1) inhibitor, 4-1BB (CD137) inhibitor, OX40 (CD134) inhibitor, B cell and T cell attenuator (BTLA) inhibitor, T cell immunoglobulin Protein inhibitors, TIM-3 inhibitors, and Killer-cell Immunoglobulin-like Receptor (KIR) inhibitors expressed on the surface of NK cells and part of T cells.
  • IDO-1 guanamine 2,3-double Oxygenase
  • 4-1BB CD137
  • OX40 CD134
  • B cell and T cell attenuator (BTLA) inhibitor B cell and T cell attenuator (BTLA) inhibitor
  • T cell immunoglobulin Protein inhibitors T cell immunoglobulin Protein inhibitors
  • TIM-3 inhibitors TIM-3 inhibitors
  • KIR Killer-cell Immunoglobulin-like
  • the immunological checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a guanamine 2,3-dual oxygenation Enzyme (IDO-1) inhibitor, 4-1BB (CD137) inhibitor, OX40 (CD134) inhibitor, B cell and T cell attenuator, T cell immunoglobulin, TIM-3, and expressed in NK cells and partial T Killer cell immunoglobulin-like receptor on the cell surface.
  • the immunological checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a guanamine 2,3-dual oxygenation Enzyme (IDO-1) inhibitor, 4-1BB (CD137) inhibitor, OX40 (CD134) inhibitor, B cell and T cell attenuator, T cell immunoglobulin, TIM-3, and expressed in NK cells and partial T Killer cell immunoglobulin-like receptor on the cell surface.
  • IDO-1
  • the domain that specifically recognizes and binds to the immune cell surface antigen PD-1 includes a light chain variable region having three CDRs (anti-PD-1 VL) and a heavy chain variable region having three CDRs (anti-PD-1) VH), wherein the light chain variable region (anti-PD-1 VL) comprises a light chain CDR (LCDR) selected from the amino acid sequences set forth in SEQ ID NOs: 1-16; and the heavy chain variable region (Anti-PD-1 VH) comprises a heavy chain CDR (HCDR) selected from the amino acid sequences set forth in SEQ ID NOS: 17-31.
  • LCDR light chain CDR
  • Anti-PD-1 VH comprises a heavy chain CDR (HCDR) selected from the amino acid sequences set forth in SEQ ID NOS: 17-31.
  • LCDR1 the amino acid sequence of which is set forth in any one of SEQ ID Nos: 1, 2, 3, 4 and 5,
  • LCDR2 the amino acid sequence of which is set forth in any one of SEQ ID Nos: 6, 7, 8, 9 and 10, and
  • LCDR3 the amino acid sequence of which is set forth in any one of SEQ ID Nos: 11, 12, 13, 14, 15 and 16;
  • the heavy chain variable region comprises:
  • HCDR1 the amino acid sequence of which is set forth in any one of SEQ ID NOs: 17, 18, 19, 20 and 21,
  • HCDR2 the amino acid sequence of which is set forth in any one of the SEQ ID NOs: 22, 23, 24, 25 and 26, and
  • HCDR3 the amino acid sequence of which is set forth in any one of SEQ ID NOs: 27, 28, 29, 30 and 31.
  • VL1 a light chain variable region VL1 comprising SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 13;
  • VL2 a light chain variable region VL2 comprising SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 12;
  • VL3 a light chain variable region VL3 comprising SEQ ID NO: 5, SEQ ID NO: 10 and SEQ ID NO: 16;
  • VL4 a light chain variable region VL4 comprising SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 11;
  • VL5 a light chain variable region VL5 comprising SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:13;
  • VL6 Light chain variable region VL6 comprising SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 14.
  • VH1 a heavy chain variable region VH1 comprising SEQ ID NO: 17, SEQ ID NO: 22 and SEQ ID NO: 27;
  • VH2 a heavy chain variable region VH2 comprising SEQ ID NO: 18, SEQ ID NO: 23 and SEQ ID NO: 30;
  • VH3 a heavy chain variable region VH3 comprising SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID NO: 29;
  • VH4 a heavy chain variable region VH4 comprising SEQ ID NO: 20, SEQ ID NO: 25 and SEQ ID NO: 30;
  • Heavy chain variable region VH5 comprising SEQ ID NO: 21, SEQ ID NO: 26 and SEQ ID NO: 31.
  • the antibody or functional fragment thereof specifically binds to an epitope located within the extracellular domain of human PD-1.
  • cytokine is selected from the group consisting of: interleukin (IL), tumor necrosis factor (TNF), interferon (IFN), colony stim ⁇ lating factor (CSF) , transforming growth factor, growth factor (GF) and chemokine family.
  • IL interleukin
  • TNF tumor necrosis factor
  • IFN interferon
  • CSF colony stim ⁇ lating factor
  • GF growth factor
  • chemokine family chemokine family.
  • colony stim ⁇ lating factor comprises G (granulocyte)-CSF, M (macrophage)-CSF, GM (granulocyte, macrophage) Cell)-CSF, Multi(multiplex)-CSF (IL-3), stem cell factor (SCF), erythropoietin (EPO);
  • the tumor necrosis factor is selected from the group consisting of TNF- ⁇ and TNF- ⁇ ;
  • the transforming growth factor is a transforming growth factor- ⁇ family (TGF- ⁇ family) TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3, TGF ⁇ 1 ⁇ 2 or bone morphogenetic protein (BMP);
  • TGF- ⁇ family transforming growth factor- ⁇ family
  • BMP bone morphogenetic protein
  • the growth factor includes epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor-I. (IGF-I), IGF-II, leukemia inhibitory factor (LIF), nerve growth factor (NGF), oncostatin M (OSM), platelet-derived endothelial cell growth factor (PDECGF), transforming growth factor- ⁇ (TGF) - ⁇ ), vascular endothelial growth factor (VEGF);
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • IGF-I insulin-like growth factor-I.
  • LIF leukemia inhibitory factor
  • NGF nerve growth factor
  • OSM oncostatin M
  • PDECGF platelet-derived endothelial cell growth factor
  • TGF transforming growth factor- ⁇
  • VEGF vascular endothelial growth factor
  • the chemokine family is selected from the group consisting of CXC/ ⁇ subfamily, CC/ ⁇ subfamily, C-type subfamily, and CX3C subfamily; wherein the CXC/ ⁇ subfamily includes IL-8, a growth-regulated oncogene / melanoma growth stimulating factor (GRO/MGSA), platelet factor-4 (PF-4), platelet basic protein (PBP/CXCL7), proteolytic-derived product CTAP-III and ⁇ -platelet globulin ( ⁇ - Thromboglobulin, ⁇ -TG), interferon-inducible protein-10, Epithelial Neutrophil-Activating Protein 78 (ENA-78); Phage inflammatory protein 1 ⁇ (MIP-1 ⁇ ), MIP-1 ⁇ , RANTES (regulated upon activation normal T-cell expressed and secreted (CCL5), monocyte chemotactic protein-1 (MCP-1/MCAF), MCP-2 MCP-3 and I-309; the C-type subfamily comprises a lymphocyte chemotactic
  • cytokine is selected from any one of IFN- ⁇ , IFN- ⁇ and IFN- ⁇ , preferably IFN- ⁇ .
  • cytokine is IFN-[gamma], preferably an IFN-[gamma] dimer.
  • the fusion according to claim 18 or 19, wherein the antibody or a functional fragment thereof comprises: the light chain CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NOs: 3, 7 and 13, respectively; The heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 21, 26 and 31, respectively; or the light chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOS: 2, 6 and 12, respectively; The heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOS: 21, 26 and 31, respectively.
  • the immunological checkpoint inhibitor is an anti-immunization checkpoint antibody, which is prepared by a phage library screening method, a hybridoma method, preferably by a phage library screening method. preparation.
  • anti-immunization checkpoint antibody is an anti-PD-1 antibody.
  • cytokine is IFN- ⁇ , preferably an IFN- ⁇ dimer.
  • An expression vector comprising the nucleic acid molecule of embodiment 25, which is selected from the group consisting of a eukaryotic expression vector and a prokaryotic expression vector.
  • a host cell comprising the expression vector of embodiment 26.
  • a method for treating a disease, ameliorating or ameliorating discomfort comprising administering the immune checkpoint inhibitor-cytokine fusion of any one of embodiments 1-20.
  • the cancer is selected from the group consisting of gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, esophageal cancer, small intestine cancer, thyroid cancer, Parathyroid carcinoma, melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer , rectal cancer, adrenal cancer, anal cancer, vulvar cancer, urethral cancer, penile cancer, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, epidermoid carcinoma, squamous cell carcinoma, Hodgkin's disease, non-Hodkin Lymphoma, endocrine system cancer, soft tissue sarcoma, central nervous system neoplasm, primary central nervous system lymphoma, spinal cord tumor, brainstem gliom
  • infectious disease is selected from the group consisting of HIV, influenza, herpes, giardiasis, malaria, leishmaniasis, or an infectious disease caused by the following viruses: Hepatitis virus (eg hepatitis A, B or C), herpes virus (eg VZV, HSV-1, HAV-6, HSV-II, CMV or Epstein's virus), adenovirus, influenza virus, vaccinia Virus, HTLV virus, dengue virus, papillomavirus, soft prion, poliovirus, rabies virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus , rotavirus, measles virus, rubella virus, parvovirus, JC virus or arbovirus viral encephalitis virus, or infectious diseases caused by bacteria: pneumococcal, mycobacteria, staphylococcus, strept
  • the disease is cancer
  • the cancer is selected from the group consisting of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glial Tumor, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, intestinal cancer, and non-small cell lung cancer
  • the infectious disease is a chronic viral infection, a bacterial infection or a parasitic infection disease, the chronic virus For HIV, HBV or HCV.
  • Figure 1 is a DNA electrophoresis pattern of colony PCR identification of the ID1 fusion protein gene.
  • Figure 2 is a SDS-PAGE electropherogram of recombinant expression of the ID1 fusion protein.
  • Lane 1 Ultrasound Broken Wall Supernatant of ID1 Fusion Protein Expression Strain; Lane 2: Ultrasound Broken Wall Supernatant of Negative Control BL21 Strain.
  • Figure 3 is a SDS-PAGE electropherogram of the ID1 fusion protein purified by affinity chromatography.
  • Lane 1 unpurified sample
  • Lane 2 unbound protein, ie, protein eluted in the effluent
  • Lane 3 eluted ID1 fusion protein.
  • Protein molecular weight standard M: Rainbow 245 broad-spectrum protein Marker (Solebao, PR1920)
  • Figure 4 shows the lysate supernatant of E. coli BL21 (DE3) cell culture transfected with pGEX-6p-1-ID1 and control E. coli BL21 (DE3) strain (BL21) treated with different dilution factors to treat HepG2- Luc's mean fluorescence intensity (MFI).
  • Figure 5 shows that purified ID1 fusion protein promotes killing of HepG2-Luc cells by CTL cells.
  • Figure 6 shows the effect of a target-to-target ratio of 50 to 1 and different concentrations of ID1 fusion protein on the efficiency of CTL killing HepG2-Luc cells. ** indicates p ⁇ 0.01; *** indicates p ⁇ 0.001.
  • Figure 7 shows the effect of in vivo imaging on the efficiency of CTL killing HepG2-Luc cells compared with 50 to 1 and different concentrations of ID1 fusion protein. The higher the fluorescence intensity, the more viable cells represent, the lower the killing efficiency. .
  • Figure 8 shows the effect of a target-to-target ratio of 10 to 1 and different concentrations of ID1 fusion protein on the efficiency of CTL killing HepG2-Luc cells. * indicates p ⁇ 0.05
  • Figure 9 shows the effect of in vivo imaging on the efficiency of CTL killing HepG2-Luc cells compared with 10 to 1 and different concentrations of ID1 fusion protein. The higher the fluorescence intensity, the more viable cells represent, the lower the killing efficiency. .
  • Figure 10 shows the effect of a target-to-target ratio of 50 to 1 and different concentrations of ID1 fusion protein on the efficiency of CTL killing THP-1 cells. **** indicates p ⁇ 0.0001
  • Figure 11 shows the purification of the tID1 fusion protein using a chromatography column Superdex 200 10/30.
  • Figure 12 shows the results of SDS-PAGE detection of the tID1 fusion protein.
  • M Rainbow 245 broad-spectrum protein Marker (Solebao, PR-1920); Lane 1 is the second tube eluate shown in Figure 11, Lane 2 is the third tube collection solution shown in Figure 11, Lane 3 is Figure 11. The fourth tube collection liquid is shown, and the lane 4 is the collected liquid after the 2-4 tubes are combined.
  • Figure 13 shows the ELISA binding curve of the tID1 fusion protein to the PD-1 protein.
  • Figure 14 shows the ELISA binding curve of tID1 fusion protein to IFNGR.
  • Panel A shows the binding curve of tID1 fusion protein to IFNGR
  • Panel B shows the binding curve of IFN- ⁇ and IFNGR.
  • Figure 15 shows the SPR assay for detecting the binding of the tID1 fusion protein to the PD1 antigen and IFNGR.
  • Figure 16 shows the ELISA binding curves of tID1 fusion protein to different species PD-1.
  • Figure 17 shows an ELISA profile of the tID1 fusion protein blocking PD-1 binding to PD-L1.
  • Figure 18 shows the binding curves of tID1 fusion protein to 293T-PD-1 cells.
  • Figure 19 shows that the tID1 fusion protein blocks the binding curve of 293T-EGFP/PD-1 to PD-L2.
  • Figure 20 shows by flow cytometry (FACS) that the tID1 fusion protein promotes tumor cell expression of PD-L1.
  • Figure 21 and Figure 22 show the inhibition of proliferation of hepG2 cells and Hela cells by tID1 fusion protein, respectively.
  • Figure 23 shows the binding of tID1 fusion protein to CD3 induced CD4+ T cells.
  • Figure 24 shows that the tID1 fusion protein promotes the killing effect of PBMC on hepG2 cells.
  • Figure 25 shows that tID1 promotes the killing effect of PBMC on human bladder cancer cell line T24.
  • a fusion refers to a structure obtained by biological genetic manipulation such as recombination or by chemical synthesis of functional structures having different mechanisms of action and targeted targets.
  • an anti-tumor cytokine with an immunological checkpoint inhibitor, more specifically, a fusion protein of IFN- ⁇ and an anti-PD-1 antibody.
  • Immunotherapy refers to the immune state of the body that is low or hyperactive, artificially enhancing or inhibiting the body's immune function to achieve treatment for disease purposes.
  • it specifically refers to immunotherapy for tumors and cancer diseases using fusions of cytokines and immunological checkpoint inhibitors, particularly tumor-inhibiting therapies using fusion proteins of IFN- ⁇ and PD-1 antibodies.
  • Tumor inhibition refers to the inhibition of the occurrence or progression of a tumor by manipulating an immunological pathway at a molecular level by a drug that specifically binds to a tumor-associated gene or a regulatory factor associated with tumor production.
  • a drug that specifically binds to a tumor-associated gene or a regulatory factor associated with tumor production specifically means that the PD-1 antibody binds to the PD-1 antigen, and the PD-1 inhibits the weakening of the T cell activity, and provides the anti-tumor activity of the T cell, thereby suppressing the growth, reproduction and slowing down of the tumor cell.
  • the purpose of tumor growth rate is not limited to the proliferation of the tumor growth rate.
  • Interferon a cytokine produced by monocytes and lymphocytes, is also a lymphokine with extensive immunomodulatory effects. It is a group of active proteins with multiple functions (mainly glycoproteins). Efficient antiviral biologically active substance. Interferon does not directly kill or inhibit the virus, but mainly causes the cells to produce antiviral proteins through the action of cell surface receptors, thereby inhibiting the replication of the virus.
  • the types are classified into three types, ⁇ -(white blood cell) type and ⁇ -( ⁇ Fibroblast type, ⁇ -(lymphocyte) type; at the same time, it can enhance the vitality of natural killer cells (NK cells), macrophages and T lymphocytes, thereby playing an immunomodulatory role and enhancing antiviral ability.
  • Lymphocyte-type interferon IFN- ⁇
  • Human IFN- ⁇ belongs to type II interferon, also known as ⁇ -IFN or immunointerferon. It is a secreted protein. It is composed of 143 amino acids after removing the signal peptide. It exists in the form of homodimer or tetramer. The molecular weight is 40 kDa.
  • IFN- ⁇ is commonly used clinically for antiviral and antitumor. IFN- ⁇ inhibits the proliferation of a variety of viruses and is widely used in the treatment of viral infections. IFN- ⁇ can inhibit tumor cell growth and prevent tumor metastasis and recurrence in vivo. Clinical application shows that IFN- ⁇ has good effects on hairy cell leukemia, melanoma, skin tumor, chronic myeloid leukemia, glioma, lymphoma and myeloma. IFN- ⁇ can directly kill tumor cells by inducing apoptosis of tumor cells and interfering with the growth cycle of tumor cells. It can also inhibit tumor growth by inhibiting the growth of blood vessels in tumor tissues, and can also enhance the body by regulating the body's immune system. The ability to remove tumor cells.
  • IFN- ⁇ activates the immune system through the following pathways: (1) activates the function of dendritic cells (DC cells), promotes the differentiation of hematopoietic cells into DC cells and the maturation of DC cells, and enhances the infiltration of DC cells into tumor sites. Enhance the antigen presentation of DC cells to T cells; (2) Activate T cells to produce specific immunity to tumor cells; at the same time, through the NF- ⁇ B pathway, T cells are protected from apoptosis and maintain long-term survival of T cells; 3) It can also be converted into macrophages by activating monocytes, which produce tumor killing effects by releasing tumor necrosis factor (such as TNF- ⁇ ).
  • TNF- ⁇ tumor necrosis factor
  • cytokines As an important cellular immune function enhancer, cytokines have been used to prepare a variety of conjugate and fusion protein drugs. However, it has not been contemplated in the art to fuse cytokines with immunological checkpoint inhibitors that have important applications in anti-tumor research. Unexpectedly, the inventors of the present application found that the fusion of the two drugs not only makes them better maintain their respective functions, but also has a synergistic effect. Therefore, fusion proteins of cytokines and immunological checkpoint inhibitors have good development and application prospects for tumor therapy.
  • Immune checkpoints are a class of immunosuppressive molecules that modulate the intensity and breadth of immune responses and prevent normal tissue from being damaged and destroyed. These "checkpoints" inhibit the immune regulatory signaling pathway, which normally inhibits the function of T cells, and may be used by tumor cells to form immune escape in tumor tissues. Therefore, in the process of tumor development and development, immune checkpoints become one of the main causes of immune tolerance.
  • Immunological checkpoint-based therapies are treatments that enhance T cell activity by co-suppressing or co-stimulating the corresponding signals to enhance anti-tumor immune responses.
  • Drugs that act on immune checkpoints have completely different characteristics in terms of anti-cancer compared to previous drugs. First, they do not directly act on tumor cells, but indirectly kill tumor cells by acting on T cells; in addition, they are not specific to the specific surface of the tumor surface, but systematically enhance the systemic anti-tumor immunity. reaction.
  • T cell immunoglobulin are all "immunoassay" molecules in tumors. Tissues may be used by tumor cells to form immune escapes, which control cell cycle progression by controlling extracellular and intracellular signals.
  • the strategy of blocking immune checkpoints such as the PD-1 pathway mainly enhances the killing effect on tumor cells from the following aspects: (1) promoting the aggregation of effector T cells at the tumor site by enhancing homing ability; (2) reducing tumor micro The number of regulatory T cells in the environment reduces its activity; (3) increases the number of effector T cells; (4) increases the cytotoxic effect of tumor-specific T cells, and the tumor-specific cytotoxic T cells reach the tumor site and pass TCR recognizes tumor cells, releases IFN- ⁇ and T cell particles to kill tumor cells to enhance killing of tumor cells; (5) enhances the production of pro-inflammatory cytokines; and (6) down-regulates potential inhibitory cytokines such as IL- 10.
  • An immunological checkpoint inhibitor of the present application refers to a structure that can bind to an immunological checkpoint and inactivate an immunological checkpoint.
  • the inhibitor may be either an organic compound that non-specifically binds to an immunological checkpoint or a ligand or antibody that specifically binds to an immunological checkpoint.
  • an immunological checkpoint inhibitor is preferably an antibody.
  • the immunological checkpoint antibody is an immunoglobulin or a functional fragment thereof, such as an antigen-binding fragment, that specifically binds to an immunological checkpoint.
  • the antibody of the present application may be a whole antibody or a single chain antibody.
  • the immunological checkpoint inhibitor of the present application may be a functional fragment of an antibody, an antigen binding fragment, such as Fab, F(ab')2, Fv, scFv, Fd or dAb.
  • the immunological checkpoint inhibitor is an anti-PD-1 antibody.
  • PD-1 is expressed on activated T cells, B cells, macrophages, and monocytes.
  • the ligand for PD-1 is the B7 family members PD-L1 (B7-H1) and PD-L2 (B7-DC). The interaction of PD-1 with its ligand can down-regulate the central and peripheral immune responses and inhibit the anti-tumor activity of T cells.
  • immunological checkpoint inhibitors which have important application prospects in anti-tumor research, are fused with cytokines to treat diseases.
  • the inventors of the present application have found that blocking the immunological checkpoint pathway can effectively perform anticancer treatment by suppressing an immune response in a tumor environment.
  • the fusion of immunological checkpoint inhibitors with cytokines not only allows them to maintain their respective functions well, but also has a synergistic effect.
  • the present application uses an prokaryotic expression system to immunological checkpoint inhibitors and IFN- in one embodiment.
  • Gamma fusion expression achieves the effect of targeting to tumor cells, such that IFN- ⁇ and immunological checkpoint inhibitors, such as anti-immunoassay antibodies, synergistically enhance anti-tumor effects; this application uses eukaryotic expression in another embodiment
  • the expression system fuses the dimeric structure of IFN- ⁇ with an immunological checkpoint inhibitor, and also achieves a synergistic anti-tumor effect.
  • the present application provides new products and uses with highly potent anti-tumor activity.
  • the present application will have an anti-immunization checkpoint antibody (in some exemplary embodiments, an anti-PD-1 antibody) with a specific sequence and a cytokine (in some exemplary embodiments, an IFN- ⁇ immunomodulatory factor)
  • the fusion forms a fusion protein as a new drug with high antitumor activity.
  • IFN- ⁇ can activate antigen presenting cells, and promote differentiation of type I helper T cells (ThI cells) by up-regulating the transcription factor T-bet.
  • ThI cells type I helper T cells
  • IFN- ⁇ can also stimulate tumor cells to enhance the expression level of PD-L1, and the PD-1/PD-L1 signal leads to inhibition of immune cell killing by tumor cells.
  • the ID1 fusion protein of the present application organically integrates the two, and can directly exert the positive tumor killing effect of IFN-W and PD-1 monoclonal antibody, and the PD-1 monoclonal antibody can effectively block the induction by IFN- Tumor cells express PD-L1-induced immunosuppression, which enhances the killing effect on tumors and compensates for their unfavorable characteristics compared with IFN-I and PD-1 monoclonal antibodies alone, further expanding the anti-tumor application.
  • the scope is expected to become a kind of artificial immunotherapy drug with stronger tumor letting ability and wider application range.
  • Example 1 Preparation of PD-1 scFv antibody with high PD-1 binding activity
  • a plurality of monoclonal candidate phage containing anti-PD-1 scFv were selected by a human phage antibody library screening method, and the phage obtained by screening was tested for binding ability to PD-1 by an ELISA method.
  • the method was as follows: ELISA plate was coated with human PD-1 recombinant protein (ACRO Biosystems, PD1-H5221) at 50 ⁇ L/well 2 ⁇ g/mL, overnight at 4 ° C; ELISA plate was blocked with 200 ⁇ L of 3% MPBS, 37 ° C for 3 h; 50 ⁇ L The selected phage antibody was added to the ELISA plate well, incubated at 37 ° C for 1.5 h; PBST was washed 3 times, and anti-M13 monoclonal antibody-HRP (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11973-MM05-50) was added, and incubated at 37 ° C for 1 h. ; TMB color developer color, A450 value after 2M sulfuric acid termination. At the same time, M13KO7 helper phage was used as a negative control.
  • human PD-1 recombinant protein ACRO Biosystems, PD1-H5221
  • the ELISA assay of Table 1 demonstrates the human anti-PD-1 scFv obtained by the phage screening method (the amino acid sequence is shown in SEQ ID NO. 32-36, and the nucleotide sequence is shown in SEQ ID NO. 42-46) It is capable of binding to PD-1 with high affinity.
  • a plurality of specific light chains are screened using a human phage antibody library screening method, comprising any of the CDRs of SEQ NO. 1-16, wherein
  • LCDR1 selected from any one of SEQ ID NOS. 1 to 5.
  • LCDR2 selected from any one of SEQ ID NOS. 6 to 10.
  • LCDR3 selected from any one of SEQ ID NOS. 11 to 16.
  • Heavy chain sequence comprises any of the HCDRs of SEQ NO. 17-31.
  • HCDR1 any one of SEQ ID NOS. 17-21
  • HCDR2 any of SEQ ID NOS. 22-26
  • HCDR3 any of SEQ ID NOS. 27-31
  • the above antibody is a fully human antibody or a murine antibody, preferably a fully human antibody.
  • Example 2 Construction and expression of a fusion protein comprising IFN- ⁇ -PD-1 scFv antibody (ID1)
  • Template human anti-PD-1 scFv sequence (the amino acid sequence of which is shown in SEQ ID NO. 32-36, the nucleotide sequence is shown in SEQ ID NO. 42-46) and truncated human IFN-
  • the ⁇ sequence (the amino acid sequence of which is shown in SEQ ID NO. 41 and the nucleotide sequence is shown in SEQ ID NO. 52) was synthesized by Jin Weizhi Biotechnology Co., Ltd.
  • pIFN-F 5'-gcggatccCAGGACCCATATGTTAAAG-3' (SEQ ID No. 37)
  • pIFN-R 5'-CTCCACCAGCTGCACCTG-3' (SEQ ID No. 38)
  • pNFV-F 5'-CAGGTGCAGCTGGTGGAG-3' (SEQ ID No. 39)
  • pNFV-R 5'-GCCTCGAGttaACGTTTGATCTCCACGTTG-3' (SEQ ID No. 40).
  • Expression vector pGEX-6p-1.
  • IFN- ⁇ and anti-PD-1ScFv were amplified by Primestar polymerase mix (TAKARA, R045A), respectively.
  • Primer F 1 ⁇ l
  • primer R 1 ⁇ l
  • template 1 ⁇ l Primestar polymerase mix 25 ⁇ l, ddH2O to 50 ⁇ l.
  • Amplification conditions 94 ° C 90 s, (94 ° C 15 s, 50 ° C 5 s, 72 ° C 10 s) ⁇ 30 cycles, 72 ° C 5 min.
  • the amplified fragment was subjected to a gel recovery kit (QIAGEN, 28704) and subjected to overlap PCR.
  • IFN- ⁇ recovery fragment 100 ng
  • anti-PD-1 scFv recovery fragment 100 ng
  • polymerase 12.5 ⁇ l supplement ddH2O to 25 ⁇ l.
  • Amplification conditions 94 ° C 90 s, (94 ° C 15 s, 50 ° C 5 s, 72 ° C 5 s) ⁇ 10 cycles, 72 ° C 5 min.
  • Reaction system pINF-F: 1 ⁇ l, pNFV-R: 1 ⁇ l, template 5 ⁇ l, Primestar polymerase mix 25 ⁇ l, supplement ddH2O to 50 ⁇ l.
  • Amplification conditions 94 ° C 90 s, (94 ° C 15 s, 55 ° C 5 s, 72 ° C 5 s) ⁇ 30 cycles, 72 ° C 5 min.
  • pGEX-6P-1 and fusion target fragment were digested with restriction endonuclease BamHI (NEB, R3136S) and XhoI (NEB, R0146S), respectively, and digested at 37 °C for 2 h, and DNA was carried out. Recycling.
  • the vector and the fragment were ligated with T4 ligase (NEB, M0202L), and the ligation system was: pGEX-6P-1 vector fragment: 1 ⁇ l, fusion target fragment: 4 ⁇ l, buffer: 1 ⁇ l, T4 ligase: 1 ⁇ l, Supplement ddH 2 O to 10 ⁇ l. After 4 h at room temperature, it was transfected into E. coli Trans-T1 competent cells (TransGen Biotech Co., Ltd., CD501). Plates were plated on 2YT medium agar plates containing a final concentration of 100 ⁇ g/ml ampicillin.
  • Seed medium (g ⁇ L -1 ): Tryptone 16, yeast powder 10, NaCl 5, pH 7.0-7.4.
  • Expression medium (g ⁇ L -1 ): Tryptone 12, yeast powder 24, glycerol 4, KH 2 PO 4 2.31, K 2 HPO 4 12.54, pH 7.0.
  • IPTG isopropyl thiogalactoside
  • Binding solution 50 mM Tris-HCl pH 7.5-8.0;
  • the column was equilibrated with 5 column volumes of binding solution; 0.5 ml/min was loaded; 5 column volumes, 1 ml/min flow rate washing column; 3-5 column volume eluent, 1 ml/min flow rate eluted to collect proteins.
  • a fusion protein having the amino acid sequence of SEQ ID No. 50 was tested for its anti-PD-1 activity.
  • Positive control 1 was set to anti-PD-1 scFv (obtained by phage expression as in Example 1), positive control 2 - human anti-PD-1 antibody (crobiosystems, anti-PD-1 mAb, Human IgG4) (concentration 2.5 ⁇ g/ml), negative control: BL21 (DE3) bacterial cell wall supernatant, sample 1 is purified ID1 protein.
  • Liquid A (3,3',5,5'-tetramethylbenzidine, TMB): 20 mg of TMB was weighed and dissolved in 10 ml of absolute ethanol, and after completely dissolved, double distilled water was added to 100 ml.
  • Results The results showed that the ID1 fusion protein has anti-PD-1 activity compared with the control group.
  • the IFN- ⁇ activity was verified by verifying that the ID1 fusion protein acts on the PD-L1 expression level of HepG2-Luc cells for a certain period of time.
  • Hepatoma cells HepG2-Luc were cultured in 24-well plates at 2 x 10 4 cells per well, 500 ⁇ l/well.
  • HepG2-Luc cells were lysed for 6-8 hours, and lysate supernatant and IFN of different concentrations of pGEX-6p-1-ID1/E.coli BL21(DE3) strain and control E.coli BL21(DE3) strain were added. - ⁇ , 3 replicate wells per concentration.
  • IFN- ⁇ mother liquor concentration 50ng/mL, working concentration 25ng/mL
  • CCK8 detection of cell proliferation inhibition was added at a volume of 1/20 of the final volume of each well, placed in a 37 ° C 5% CO 2 incubator for 1 h, and the corresponding well supernatant was taken up to 100 ⁇ L to a new 96-well plate, and the detection was performed at a wavelength of 450 nm.
  • the OD of the ID1 fusion protein and the positive control and the negative control solution; the cells of each well were digested and collected in a 1.5 mL centrifuge tube, and the PD-L1 expression level of HepG2-Luc cells was detected by flow cytometry.
  • PBMC Peripheral blood mononuclear cells
  • step (3) Take a 50mL centrifuge tube, first add the same volume of human peripheral blood lymphocyte separation solution (Ficoll, Tianjin Haoyang Biological Products Technology Co., Ltd., LTS1077) with the diluted cells of step (2), and then use a straw Carefully pipette the cells onto the Ficoll level.
  • human peripheral blood lymphocyte separation solution Ficoll, Tianjin Haoyang Biological Products Technology Co., Ltd., LTS1077
  • Mitomycin C treatment of HepG2-Luc and THP-1 cells Resolving mitomycin C in serum-free medium (sigma, M0503) at a working concentration of 10 ⁇ g/mL at 37 ° C 5% CO 2 Incubate in the incubator, and incubate for 1.5 h every 10 min; collect the stimulated HepG2 and THP-1 cells, and wash them with PBS three times to remove residual mitomycin C as a stimulating cell.
  • the cells co-cultured in the step 2 were collected, that is, specific CTL cells, washed twice with PBS, counted, and the cell concentration was adjusted.
  • HepG2-Luc and THP-1 cells (1 ⁇ 10 4 /well) were mixed with specific CTL in a ratio of 50:1 or 10:1, and grouped according to Table 2, Purified ID1 fusion protein samples and controls, total volume 200 ⁇ L, 3 replicate wells in each group.
  • the target cells were THP-1 using a lactate dehydrogenase (LDH) cytotoxicity test kit (Biyuntian, C0016), and the absorbance was measured at 490 nm, and the killing efficiency of each group of samples and the control substance against the cells was statistically analyzed;
  • LDH lactate dehydrogenase
  • the target cells were HepG2-Luc group, and 100 ⁇ L of D-luciferin (0.075 mg/mL) (MCE, HY-12591A) was added to the wells of the remaining 100 ⁇ L medium for 5 min in the dark, and then detected by a living imager (spectral Amix). HepG2-Luc (with Fg-Luc fluorescent gene) was used to measure the fluorescence intensity of cells, and the cell killing efficiency of each group was statistically analyzed.
  • the purified ID1 fusion protein promotes the killing effect of CTL cells on HepG2-Luc cells as shown in FIG.
  • the effect of different concentrations of ID1 fusion protein on the efficiency of CTL killing HepG2-Luc cells was determined by detecting the effective target ratios of 50:1 and 10:1, respectively. The results are shown in Fig. 6 (** indicates p ⁇ 0.01; *** indicates p ⁇ 0.001) and Figure 8 (* indicates p ⁇ 0.05).
  • In vivo imaging was used to detect the effect of different concentrations of ID1 fusion protein on the efficiency of CTL killing HepG2-Luc cells at 50:1 and 10:1.
  • the results are shown in Figure 7 and Figure 9, where the fluorescence intensity is higher. High, representing more living cells, the lower the killing efficiency.
  • the effect of the ID1 fusion protein on the efficiency of CTL killing of THP-1 cells at 50:1 was shown in Figure 10 (**** indicates p ⁇ 0.0001).
  • the ID1 fusion protein of the present application can promote the killing effect of specific CTL on HepG2-Luc and THP-1, and the experimental group added with the ID1 fusion protein has significant difference compared with the positive control.
  • Example 6 Construction of eukaryotic expression vector pBudCE4.1 containing the tID1 fusion protein gene of (IFN- ⁇ ) 2 -PD-1 scFv2
  • SEQ ID No: 36 which comprises the light chain CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NOs: 3, 7 and 13, respectively, and SEQ ID NO: 21, respectively.
  • SEQ ID NO: 60 containing the (IFN- ⁇ )2-PD-1 scFv2 gene.
  • Example 7 Eukaryotic expression and purification of tID1 fusion protein
  • the pBudCE4.1-tID1 construct was electroporated into 293T/17 cells, and after 7 days, the cell culture medium was collected to obtain a supernatant expressing the tID1 fusion protein (with a His purification tag).
  • Ni column (GE Healthcare Ni Sepharose TM excel, GE Cat. No. 45-003-011) was equilibrated with an equilibration solution of 20 column volumes;
  • the collected fusion protein expression supernatant was applied at 0.5 ml/min; the Ni column was washed at a flow rate of 1 ml/min using 5 column volumes of the washing solution;
  • the Ni column was eluted at a flow rate of 1 ml/min using 3-5 column volumes of eluate to collect the tID1 fusion protein;
  • the eluate was concentrated using an ultrafiltration centrifuge tube to obtain a tID1 fusion protein at a concentration of 8.9 mg/mL.
  • Example 8 tID1 fusion protein fusion protein blocks PD-1 binding to PD-L1
  • the detection OD value was detected at a wavelength of 450 nm, and for each tID1 fusion protein concentration, three duplicate wells were set.
  • the antigen IFNGR (Fc-tag) was coated on the ELISA plate at 2.5 ug/mL and 100 ⁇ L/pack;
  • the tID1 fusion protein (0.257 mg/mL purity 98%) was diluted according to the following 3A series; the positive control IFN- ⁇ was serially diluted according to the following Table 3B;
  • tID1 fusion protein and the positive control were added to the corresponding wells of the ELISA plate at 100 ⁇ L/well, and incubated at 37 ° C for 1.5 h;
  • APC-anti human IFNG Ab (1:400), 100 ⁇ L / well, incubate at 37 ° C for 1 h;
  • the detection OD value was detected at a wavelength of 450 nm, and for each tID1 fusion protein concentration, three duplicate wells were set.
  • Figure 14 The binding curve is shown in Figure 14, in which Figure A is the binding curve of tID1 fusion protein to IFNGR, and Figure B is the binding curve of IFN- ⁇ and IFNGR.
  • the ka (1/(M*s)) of the tID1 fusion protein bound to the PD-1 antigen was 8.17e+05
  • Kd(1/s) was 5.17e-03
  • KD(M) was 6.32.
  • positive control anti-PD-1 antibody (Pdab) binding to PD-1 antigen ka (1/(M*s)) was 3.88e+05
  • Kd(1/s) was 1.21e-03
  • KD (M) is 3.11e-09
  • ka(1/(M*s)) of tID1 fusion protein binding to IFN- ⁇ receptor is 2.04e+06
  • Kd(1/s) is 1.64e-03
  • KD (M) is 8.05e-10.
  • the above data indicate that the tID1 fusion protein has a good affinity with the PD-1 antigen and the IFN- ⁇ receptor, and can meet the therapeutic needs.
  • ELISA was used to detect the binding of tID1 fusion protein to human, mouse, dog and monkey PD-1.
  • the antigen human PD-1 mouse PD-1, canine PD-1, and monkey PD-1 were coated with ELISA plate at a concentration of 2 ug/mL, respectively.
  • tID1 fusion protein (initial concentration: 0.257 mg/mL, purity 98%) was diluted to different concentrations according to the following Table 4, and added to the ELISA plate at 100 ⁇ L/well, and incubated at 37 ° C for 1.5 h.
  • the tID1 fusion protein binds to human PD-1 and monkey PD-1, and does not bind to both mouse PD-1 and canine PD-1, thereby showing that the fusion tID1 fusion protein obtained by the present invention is human PD- 1 specific.
  • the 293T-PD-1 cell line is a cell line constructed by the applicant to stably express the PD-1 antigen, and the 293T cell line used for construction is ATCC product number CRL-11268.
  • Table 6 tID1 fusion protein to bind EC 293T-PD-1 cells in 50
  • the PD-L2 used in the method was HumanB7-DC/CD273protain-His (Cat: 10292-H08H, SinoBiological). Detection was performed by flow cytometry.
  • the binding curve of the tID1 fusion protein blocking 293T-EGFP/PD-1 to PD-L2 is shown in FIG.
  • Example 9 tID1 fusion protein promotes tumor cell expression of PD-L1
  • Hepatoma cells hepG2 and cervical cancer hela were seeded in 24-well culture plates at appropriate densities, respectively.
  • step 3 Replace the cells in the 24-well plate of step 1 with a new complete medium, 500 ⁇ L/well.
  • APC-CD274 antibody APC anti-human CD274 (PDL1, B7H1) Antibody APC, eBioscience) was added and incubated on ice for 30 min in the dark.
  • PD-L1 expression increased with increasing concentration of tID1 fusion protein, but when the concentration of tID1 fusion protein increased above 2.72 nM, PD-L1 expression level reached saturation and no longer increased (Fig. A);
  • the tID1 fusion protein at a lower concentration of 0.34 nM resulted in a higher level of hela cells than the positive control IFN- ⁇ PD-L1 (see Figure B), but did not increase PD- as the concentration continued to increase.
  • the hepatoma cell hepG2 and the cervical cancer cell hela can be more effectively promoted to express PD-L1 when it is lower than the same molar IFN- ⁇ , which indicates that the tID1 fusion protein has better targeting specificity.
  • the data in Figure 1 also showed that the tID1 fusion protein promoted the different concentrations of hepatoma cell hepG2 and cervical cancer cell hela expression in PD-L1 equivalent to the IFN- ⁇ positive control, the former being 2.72 nM, the latter being lower than the latter 0.34 nM, which also provides a basis for subsequent screening of drug concentrations in different tumor cell lines.
  • Example 9 Effect of tID1 fusion protein on tumor cell proliferation
  • tID1 fusion protein The effect of tID1 fusion protein on tumor cell proliferation was tested using hepatoma cell hepG2 and cervical cancer cell hela. Methods as below:
  • Hepatoma cells hepG2 and cervical cancer cells hela were inoculated separately into 96-well culture plates at appropriate densities.
  • the cell proliferation assay was performed using the enhanced CCK8 assay kit (Biyuntian Biotechnology Co., Ltd., Cat. No. C0041).
  • Binding of the tID1 fusion protein to CD3-induced CD4+ T cells was detected by flow cytometry. Methods as below:
  • lymphocyte separation solution Ficoll separation of human peripheral blood mononuclear cells PBMC.
  • CD4+ T cells were induced and cultured by adding 50 ng/mL of CD3 and 100 U/mL of IL-2 to PBMC cells.
  • Two-step CD4+ T cells were seeded at 3 x 10 5 /50 ⁇ L into V-type 96-well plates.
  • the cells of each well were resuspended in 100 ⁇ L of PBS, transferred to a 1.5 mL centrifuge tube, and the binding of the tID1 fusion protein to CD3-induced CD4+ T cells was detected on a flow cytometer.
  • tID1 fusion protein promotes killing of target cells by PBMC
  • PBMC cells were added to a round-bottom 96-well plate at 50 ⁇ L/well, and different concentrations of tID1 fusion protein were added according to the experiment, and 50 U/mL of IL-2 was added for stimulation, and the final volume of each well was adjusted to 100 ⁇ L;
  • HepG2-luc cells were added to a round-bottom 96-well plate at 50 ⁇ L/well, and different concentrations of tID1 fusion protein samples were also added according to the experiment, and the final volume of each well was adjusted to 100 ⁇ L;
  • steps 3) and 4) were each placed in a 37 ° C incubator for 10 min;
  • step 6) Move the HepG2-luc cells of step 4) to the PBMC of step 3), mix well, prepare a killing hole with a target ratio of 50:1, and a final volume of 200 ⁇ L per well, and incubate in a 37 ° C incubator;
  • the tID1 fusion protein was incubated with PBMC and hepatocellular carcinoma cell hepG2 at three concentrations of 1.02 nM, 10.2 nM and 102 nM for 16 h and 24 h.
  • the results showed that: with anti-PD1 antibody: Anti-PD1 mAb, Human (IgG4) Lot No.
  • the method is as described in the experiment (I) of the present embodiment.
  • the human bladder cancer cell line T24 cell line was purchased from ATCC article number HTB-4. See Figure 25 for the results.
  • the tID1 fusion protein was incubated with PBMC and human bladder cancer cell line T24 for 24 h at both concentrations of 10.2 nM and 102 nM.
  • the results showed that the 10.2 nM tID1 fusion protein was stronger than the same molar positive control (anti-PD1 antibody, sourced from above).
  • the PBMC promoted the killing activity of human bladder cancer cell line T24, and it was statistically significant.
  • the immunotherapeutic product provided by the present application has both the tumor killing effect against tumor cytokines and the precise targeting advantage of immunological checkpoint inhibitors. Therefore, the fusion of cytokines and immunological checkpoint inhibitors not only makes them better maintain their respective functions, but also has a synergistic effect, so that the fusion of the present application is a new tumor treatment plan with important development prospects in the field of tumor treatment.
  • the fusion comprising the immunological checkpoint inhibitor and the cytokine or the protein or polypeptide comprising the same provided by the embodiments of the present application can effectively treat the disease, improve or alleviate the discomfort, and is suitable for the development and industrial production of the new drug.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Rheumatology (AREA)
  • Pain & Pain Management (AREA)
  • Mycology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un hybridome comprenant un inhibiteur de point de contrôle immunitaire et des cytokines, des molécules d'acide nucléique codant pour l'hybridome, un vecteur d'expression correspondant, une cellule hôte, et un procédé comprenant la préparation de l'hybridome. La présente invention concerne également un procédé de détection de l'activité de l'hybridome et un procédé d'utilisation de l'hybridome dans le traitement d'une maladie et de l'amélioration ou du soulagement d'une sensation gênante.
PCT/CN2018/115286 2017-11-14 2018-11-13 Hybridome comprenant un inhibiteur de point de contrôle immunitaire, procédé de préparation de celui-ci, et applications associées Ceased WO2019096137A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201711123036 2017-11-14
CN201711123036.9 2017-11-14

Publications (1)

Publication Number Publication Date
WO2019096137A1 true WO2019096137A1 (fr) 2019-05-23

Family

ID=65748366

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/115286 Ceased WO2019096137A1 (fr) 2017-11-14 2018-11-13 Hybridome comprenant un inhibiteur de point de contrôle immunitaire, procédé de préparation de celui-ci, et applications associées

Country Status (2)

Country Link
CN (1) CN109503718B (fr)
WO (1) WO2019096137A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109705211B (zh) 2017-10-26 2020-08-18 苏州复融生物技术有限公司 一种IgG1 Fc单体及其应用
AU2020255706B2 (en) * 2019-03-29 2025-02-27 Taipei Medical University Porous hollow fiber membrane and methods of using it to select immune checkpoint inhibitor
CN117731760A (zh) * 2020-03-09 2024-03-22 四川大学华西医院 IFN-γ在制备抗肿瘤辅助药物中的应用
CN112341538A (zh) * 2020-10-27 2021-02-09 苏州复融生物技术有限公司 一种Fc单体多肽及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104479019A (zh) * 2014-12-26 2015-04-01 上海复宏汉霖生物技术有限公司 一种抗ctla-4人源抗体
CN104479020A (zh) * 2014-12-26 2015-04-01 上海复宏汉霖生物技术有限公司 一种抗pd-1人源抗体
CN105263521A (zh) * 2014-01-15 2016-01-20 卡德门企业有限公司 免疫调节剂
CN108250303A (zh) * 2016-12-19 2018-07-06 南京金斯瑞生物科技有限公司 单域抗体融合蛋白及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103536917B (zh) * 2013-10-30 2015-03-11 苏州丁孚靶点生物技术有限公司 干扰素在治疗肿瘤中的用途及相关的产品和方法
JP2018525438A (ja) * 2015-07-22 2018-09-06 エイチゼットエヌピー リミテッド がんの処置における免疫調節剤とｐｄ−１またはｐｄ−ｌ１チェックポイント阻害剤との組合せ
CN107082812B (zh) * 2017-03-29 2018-11-13 上海科医联创生物科技有限公司 一种恢复衰竭性免疫细胞功能的融合蛋白及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105263521A (zh) * 2014-01-15 2016-01-20 卡德门企业有限公司 免疫调节剂
CN104479019A (zh) * 2014-12-26 2015-04-01 上海复宏汉霖生物技术有限公司 一种抗ctla-4人源抗体
CN104479020A (zh) * 2014-12-26 2015-04-01 上海复宏汉霖生物技术有限公司 一种抗pd-1人源抗体
CN108250303A (zh) * 2016-12-19 2018-07-06 南京金斯瑞生物科技有限公司 单域抗体融合蛋白及其应用

Also Published As

Publication number Publication date
CN109503718B (zh) 2020-02-21
CN109503718A (zh) 2019-03-22

Similar Documents

Publication Publication Date Title
JP7746499B2 (ja) Ｃｌｅｃ９ａ結合物質およびその使用
EP3348571B1 (fr) Anticorps à domaine unique pour le ligand du récepteur de mort cellulaire programmée (pd-l1) et protéine dérivée de celui-ci
CN108883180B (zh) Clec9a结合剂及其用途
KR102877915B1 (ko) 섬유아세포 결합제 및 이의 용도
US10800828B2 (en) Switchable non-scFv chimeric receptors, switches, and methods of use thereof to treat cancer
JP2019001789A (ja) ヒトｃｓｆ−１ｒに対する抗体とヒトｐｄ−ｌ１に対する抗体の併用療法
EP3664829A1 (fr) Agents de liaison à pd-1 et pd-l1
TW201843171A (zh) 在酸性pH結合至VISTA之抗體
WO2019096137A1 (fr) Hybridome comprenant un inhibiteur de point de contrôle immunitaire, procédé de préparation de celui-ci, et applications associées
JP2023545057A (ja) 抗デクチン－１抗体及びその使用方法
JP2023554097A (ja) 二機能性抗ｐｄ１／ｉｌ－７分子
WO2019096136A1 (fr) Anticorps anti-pd-1, son procédé de préparation et son utilisation
JP2025525886A (ja) Ｃｄ２８に対する多機能性分子
JP2023506306A (ja) Ｉｌ－７バリアントを含む二機能性分子
CN117813320A (zh) 嵌合蛋白和免疫治疗方法
JP2024515263A (ja) 改善された特性を有する二機能性分子のための新規足場構造
WO2025242835A1 (fr) Molécules comprenant des lieurs de masquage et leurs utilisations pour le traitement du cancer
WO2023025187A1 (fr) Anticorps se liant de manière spécifique à cd47, virus oncolytique recombinant de celui-ci et son utilisation
BR122025000049A2 (pt) Agentes de ligação a clec9a e uso dos mesmos

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18879666

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18879666

Country of ref document: EP

Kind code of ref document: A1