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WO2019066536A1 - Nouvelle composition pour le traitement du cancer - Google Patents

Nouvelle composition pour le traitement du cancer Download PDF

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WO2019066536A1
WO2019066536A1 PCT/KR2018/011505 KR2018011505W WO2019066536A1 WO 2019066536 A1 WO2019066536 A1 WO 2019066536A1 KR 2018011505 W KR2018011505 W KR 2018011505W WO 2019066536 A1 WO2019066536 A1 WO 2019066536A1
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protein
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cancer
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cells
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김윤경
남기훈
양유수
김인산
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Korea Institute of Science and Technology KIST
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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
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to a novel composition for treating cancer.
  • Methods for treating cancer include surgery, radiation therapy, and chemotherapy. However, these treatments may be accompanied by side effects or limited treatment depending on cancer progression.
  • anti-cancer drugs have increased in quantity in terms of repeated research, but there has been no significant change in terms of quality. The reason for this is that most of the anticancer drugs act as a mechanism to stop and kill the cell cycle of intact cells. In addition to the cancer cells, they also attack normal dividing cells and cause side effects such as hair loss, anorexia and leukocytosis And a decrease in immunity.
  • Doxorubicin a representative anticancer drug, is an anticancer agent belonging to an anthracycline antitumor agent.
  • Anthracycline anticancer agent is an anticancer agent that selectively acts on the cell cycle, inhibits cell division, and is useful for the treatment of malignant lymphoma (lymphoma, Hodgkin's disease and non- It is used to treat various cancers such as gastrointestinal cancer (gastric cancer, liver cancer, rectum cancer, gall bladder cancer, gall bladder cancer, colon cancer, pancreatic cancer), acute myelogenous leukemia, soft tissue osteosarcoma, breast cancer, ovarian cancer, lung cancer, Recent studies have reported that an anthracycline anticancer drug induces preapoptotic translocation of caleticulin to the cell membrane leading to immunological cell death of cancer cells (Obeid et al . , Nat. Med.
  • the present invention aims at solving various problems including the above problems and provides an immunotherapeutic agent capable of maximizing the efficiency of cancer immunotherapy through induction of immunogenic cell death and control of immune cell networking and its use The purpose.
  • these problems are exemplary and do not limit the scope of the present invention.
  • a pharmaceutical composition for treating cancer comprising, as an active ingredient, a signal-regulatory protein or a fusion protein comprising the signal-regulating protein and an immunological cell death-inducing agent.
  • a signal-regulatory protein or a fusion protein comprising the signal-regulating protein and an immunogenic apoptosis-inducing agent in the manufacture of a pharmaceutical composition for the treatment of cancer .
  • a method of treating a subject comprising administering to a subject in need of treatment a signal-regulatory protein or a fusion protein comprising said signal-regulating protein and an immunological inducing agent Or a pharmaceutically acceptable salt thereof.
  • the therapeutic effect of cancer can be maximized by the synergistic effect of the combined administration of the signal regulatory protein and the immunogenic proliferation inducer.
  • FIG. 1A is a schematic diagram showing a schematic view of a doxorubicin-loaded Sirp protein multimer loaded with doxorubicin inside a nanocage produced by self-assembly of a fusion protein composed of ferritin heavy chain protein and Sirp alpha according to an embodiment of the present invention
  • FIG. 1B is a graph showing fluorescence FPLC analysis of FHSirp alpha-dox loaded with doxorubicin according to an embodiment of the present invention.
  • FIG. FIG. 1D is a histogram showing the results of analysis of the particle size of FHSirp alpha-dox loaded with doxorubicin by dynamic light scattering (DLS) analysis.
  • FIG. 1D is a histogram showing FHSirp alpha-dox loaded with doxorubicin It is a photograph taken with a transmission electron microscope.
  • FIG. 2A is a schematic diagram showing an experimental schedule of an animal experiment for analysis of in vivo anticancer activity of Experimental Example 1-1
  • FIG. 2B is a graph showing the results of in vivo anticancer effect analysis performed according to the schedule of FIG. P ⁇ 0.05, ***: P ⁇ 0.001).
  • FIG. 2C is a photograph of the tumor area of each experimental group on the 25th day after inoculation of cancer cells (left and right are independent experimental animals in the same experimental group) (***: P ⁇ 0.001) showing the result of measuring the weight of the tumor tissue extracted from each experimental group.
  • FIG. 3A is a graph showing the presence and proportion of macrophages and dendritic cells in tumor tissues extracted from FHSirp? -Dox according to an embodiment of the present invention and a cancer model animal to which a buffer was administered as a control group. (Left) and CD11c-positive cells (right) as compared to the total number of cells isolated from tumor tissues, and FIG. 3B is a histogram showing the results of FACS analysis using anti-CD11c antibody (*: P ⁇ 0.05, ***: P ⁇ 0.001).
  • FIG. 4a shows the effect of various doses of CT26.CL25 on the tumor drained lymph node cells after administration of a buffer or various experimental materials (dox, wrFH-dox, mSirp ⁇ + > dox and FHSirp & (*: P ⁇ 0.05, **: P ⁇ 0.01).
  • FIG. 4B is a graph showing the results of measurement of the expression level of INF- ⁇ upon treatment of peptides ( ⁇ -galactosidase, AH1 peptide and PIA peptide derived from gp70) (*: P ⁇ 0.05, **: P ⁇ 0.01) in the splenocytes isolated from the experimental animals of FIG. 4A.
  • FIG. 5 is a graph showing the results of comparing the accumulation level of CD8 + T cells in the tumor site after the administration of the buffer solution or the combined administration of doxorubicin (FHSirp ⁇ -dox) according to one embodiment of the present invention.
  • the upper part is a fluorescence staining image of a tumor tissue section after the buffer solution administration
  • the lower part is a fluorescence staining image of a tumor tissue section after administration of FHSirp? -Dox according to one embodiment of the present invention
  • the second column on the left shows the result of staining with anti-CD8 antibody.
  • the third column shows the result of the DAPI staining and the anti-CD8 antibody staining result
  • the rightmost column shows the result of cell segmentation analysis.
  • FIG. 6A shows the results of a single dose of the buffer solution, wtFH-dox, and FHSirp? -Dox according to one embodiment of the present invention in a tumor model by the CT26.CL25 cancer cell inoculation and measuring the size of the tumor over time is a graph showing the results (**: P ⁇ 0.01, I ***: P ⁇ 0.001), Figure 6b is a picture taken with the tumor inoculation site of the cancer in the animal model, 25 days after tumor inoculation, Figure 6c is a (*: P ⁇ 0.05, **: P ⁇ 0.01) obtained by measuring the weight of the tumor tissues extracted from the experimental animals of FIG. 6A on the 25th day after inoculation of cancer cells.
  • FIG. 7A is a graph showing the results of tumor necrosis after 25 days of inoculation of cancer cells after administration of a buffer solution or various experimental materials (dox, wtFH-dox, mSirp ⁇ + dox and FHSirp ⁇ -dox) to cancer model animals by CT26.CL25 cancer cell inoculation After suturing, 1 ⁇ 10 6 cells of the same amount as that of CT26.CL25 tumor cells inoculated on the opposite side were inoculated and rechallenged. After that, the control and FHSirp ⁇ -dox treatment groups were treated with the second cancer cell inoculation site over time
  • FIG. 7B is a graph showing the ratio of tumor-free animals according to the passage of time of the animal model animal
  • FIG. FIG. 7C is a graph showing the survival rate of the animal model animal over time
  • FIG. 8A shows the results of the intravenous injection of a buffer solution or various experimental substances (dox, wtFH-dox, mSirp ⁇ + dox and FHSirp ⁇ -dox) to cancer model animals by CT26 cancer cell inoculation, a graph showing the result of measuring the size (***: P ⁇ 0.001)
  • Fig. 8b is a graph illustrating results of measuring the weight of the excised tumor tissue 25 days after tumor inoculation in the tumor model animals (*: P ≪ 0.05, ***: P ⁇ 0.001).
  • FIG. 9a shows the results of the intravenous injection of a buffer solution or various experimental substances (dox, wtFH-dox, mSirp ⁇ + dox and FHSirpirp ⁇ -dox) to female model animals by B16F10-Ova cancer cell inoculation
  • FIG. 9B is a graph showing a result of measuring the weight of tumor tissue extracted on the 25th day after inoculation of cancer cells in the animal model animal (** ( P : : P ⁇ 0.01).
  • FIG. 10 is a graph showing the results of single-cell culture of tumor cells after administration of a buffer solution or FHSirp? -Dox to a cancer model animal by inoculation with B16F10-Ova cancer cells, and dendritic cells isolated therefrom are co-cultured with OT-1 T cells, (*: P ⁇ 0.05) by determining the amount of INF-y present.
  • FIG. 11A shows the results obtained by injecting a buffer solution or various experimental substances (dox, FHSirp ⁇ , and FHSirp ⁇ -dox) into animal models of cancer by the B16F10.Ova cancer cell inoculation and injecting CFSE-stained OT-1 T-
  • FIG. 11B is a histogram showing the results of FACS analysis after single cellization of the extracted tumor-draining lymph node cells
  • FIG. 11B is a histogram showing the percentage of the generation of OT-1 T-cells stained with CFSE (**: P ⁇ 0.01).
  • FIG. 12A is a series of fluorescence micrographs that analyze the effect of BMDM of FHSirp ⁇ and FcSirp ⁇ on cancer cells (HT29) according to an embodiment of the present invention
  • FIG. 12B is a photograph showing the cancer cell phagocytosis rate
  • Figure 12C is a graph showing the results of quantification (*: P ⁇ 0.05; ***: P ⁇ 0.001) of FcSirp alpha and FHSirp alpha at various concentrations (50 nM, 500 nM, and 5 ⁇ M) (***: P ⁇ 0.001).
  • FIG. 13A is a graph showing the effect of FcSirp alpha and / or mitoxantrone according to an embodiment of the present invention on C57BL / 6 wild-type mice subcutaneously injecting B16F10 cancer cells and inducing cancer
  • FIG. 13B is a graph showing the survival rate of the experimental animals used in the experiment of FIG. 13A until the 21st day after the injection of the cancer cells (FIG. 13B)
  • FIG. 13C is a graph showing the results of measurement of the average weight of cancer tissues obtained after sacrifice of the experimental animals on day 21 after the injection of cancer cells
  • FIG. 13D is a graph showing the change in body weight of the experimental animals And the results are shown in FIG.
  • FIG. 14 is a graph showing the results of the administration of FcSirp alpha and / or mitoxantrone according to one embodiment of the present invention to C57BL / 6 wild-type mice subcutaneously injecting B16F10 cancer cells and inducing cancer, CD8 + T cell infiltration by performing flow cytometry analysis using an anti-CD8 antibody after monoclonalization of the tumor tissue after sacrificing the tumor cells (*: P ⁇ 0.05).
  • the term " immunogenic cell death" refers to a cell proliferation inhibitor such as anthracyclines, oxaliplatin and bortezomib, or a type of cell growth inhibitor induced by radiotherapy and photodynamic therapy Cell death.
  • a cell proliferation inhibitor such as anthracyclines, oxaliplatin and bortezomib
  • the immunological apoptosis of cancer cells can induce an effective anti-cancer immune response through activation of dendritic cells and thus activation of specific T cell responses.
  • the substance causing immunogenic cell death is called " immunogenic cell death inducer ". Immunogenic and immunogenic inducers of apoptosis are well described in Kroemer et al . ( Annu. Rev. Immunol ., 31: 51-72, 2013). This document is incorporated herein by reference in its entirety.
  • multimerization domain refers to a protein domain that contains minimal sites that play an important role in homologous recombination of homologous proteins.
  • Such multimerization domains include self-assembling domains of various self-assembling proteins such as dimerization, trimerization, tetramerization, hexamerization, 12merization, and 24merization domains.
  • the dimerization domain specifically includes an Fc fragment comprising the hinge region of the heavy chain of the antibody and the CH2 and CH3 moieties, the cytoplasmic domain of the receptor tyrosine kinase (RTK), the dimerization domain of kinesin, the dimerization domain of fibronectin, the Tol- (TLR) dimerization domains, tubulin dimerization domains, and the like.
  • the trimerization domain includes a trimerization domain of collagen, a trimerization domain of TRAIL, a trimerization domain of Eml4 protein, a trimerization domain of Clathrin, and the like.
  • the quaternization domain of p53, the quaternization domain of DsRed, and the quaternization domain of acetylcholine esterase (AChE) exist in the above-described quaternization domain.
  • the hexamerization domain includes the hexamerization domain of the HSP100 protein, the 12merization domain includes SPD (surfactant protein D, WO2013115608A1) and M. tuberoculosis Hsp16.3.
  • the 24- merization domain includes ferritin heavy chain protein, ferritin light chain protein, M. jannanschii Hsp16.5, and yeast Hsp26.
  • self-assembled proteins &quot proteins that are mass-produced by self-assembly are referred to as " self-assembled proteins ", which self-assembled proteins form multimers by regular arrangement at the same time as they are expressed without the aid of special inducers, Or a protein that can be formed.
  • Self-assembling proteins include sHsp (small heat shock protein), ferritin, vault, P6HRC1-SAPN, M2e-SAPN, MPER-SAPN, and various virus or bacteriophage capsid proteins.
  • the self-assembled proteins are well described in Hosseinkhani et al. ( Chem. Rev. , 113 (7): 4837-4861, 2013). This document is incorporated herein by reference in its entirety.
  • Sirp signal-regulated protein
  • CD47 protein the broadly expressed transmembrane protein
  • CD47 protein the broadly expressed transmembrane protein
  • innate immune cells such as host cell phagocytosis.
  • This is similar to the self-signal provided by MHC I family molecules via Ig-like or Ly49 receptors.
  • Cancer cells overexpressing CD47 activate Sirp or Sirp gamma to inhibit macrophage-mediated destruction. Recent studies have shown that high-affinity mutants of Sirp ⁇ increase the phagocytosis of cancer cells by masking CD47 on cancer cells (Weiskopf et al ., Science 341 (6141): 88-91, 2013).
  • ferritin heavy chain protein &quot refers to a protein that constitutes the heavy chain subunit of ferritin, a major intracellular iron storage protein in prokaryotes and eukaryotes , And the ferritin protein consists of 24 subunits of the ferritin heavy chain and the light chain, respectively.
  • the main function of ferritin proteins is to store iron in a water-soluble, non-toxic state.
  • the ferritin heavy chain protein has been known to self-assemble into 24 subunits without light chain protein to form hollow internal nanoparticles (Cho et al ., Biochem. Biophys. Res. Commun . 327 (2): 604-608, 2005) .
  • the ferritin heavy chain protein can act as a nanocage by loading other drugs into the empty internal space of self-assembled nanoparticles, and due to these properties, is being studied for the purpose of drug delivery.
  • anthracycline-type anticancer agent refers to an antracycline-type anticancer agent, such as Streptomyces peucetius var. refers to a cell cycle non-specific anticancer agent family used in cancer chemotherapy derived from caesius.
  • Anthracycline-based anticancer agents are used for the treatment of various cancers including leukemia, lymphoma, breast cancer, stomach cancer, uterine cancer, ovarian cancer, bladder cancer and lung cancer.
  • the first anthracycline anticancer drugs discovered were daunorubicin, followed by doxorubicin, followed by epirubicin, idarubicin, and pixantrone.
  • anthracycline-based anticancer agents is to inhibit DNA and RNA synthesis by intercalating between base pairs of DNA / RNA strands, thereby inhibiting the replication of rapidly growing cancer cells, inhibiting the activity of topoisomerase II enzyme, Inhibiting transcription and replication by inhibiting DNA relaxation, inducing DNA damage, protein and cell membrane damage and DNA damage through the formation of iron-mediated free oxygen radicals, chromatin to deregulate epigenomes and transcripts And induction of histone excretion. Recent studies have shown that doxorubicin increases the Th1 immune response by activating CD4 + cells (Park et al ., Int. Immunopharmacol .
  • dendritic cells have been reported to exhibit anticancer activity by inducing immunogenic cell death of osteosarcoma in combination with doxorubicin (Kawano et al ., Oncol. Lett . 11: 2169-2175, 2016).
  • taxanoid anticancer agent or " taxane anticancer drug " as used herein refers to diterpenoid taxane derivatives derived from Taxus sp. It is a mitotic inhibitor with a mechanism of promoting assembly and inhibiting disassembly.
  • paclitaxel and docetaxel are presently available. Among them, paclitaxel is a taxane-based chemotherapeutic agent extracted from juniper of Taxus brevifolia .
  • Docetaxel is a taxane-based anticancer drug derived from Taxus bacaata , which has similar efficacy to paclitaxel and is used for the treatment of breast cancer, non-cell lung cancer, lymphoma, bladder cancer and the like, and is more hydrophilic than paclitaxel.
  • Taxus bacaata Taxus bacaata
  • the taxane-based anticancer agent also has a mechanism of promoting immunological cell death of these cancer cells by sensitizing cancer cells to cytotoxic T lymphocytes.
  • immune checkpoint inhibitor refers to a type of drug that blocks certain types of immune system cells, such as T lymphocytes, and certain proteins produced by some cancer cells, And prevents T lymphocytes from killing cancer cells. Thus, when these proteins are blocked, the "braking device" of the immune system is released and T lymphocytes can kill cancer cells better.
  • PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2 are well known as the above-mentioned "Immune Check Point".
  • PD-1 inhibitors include Pembrolizumab (trademark: Keytruda), Nivolumab (trade name: Opdivo), PD-1 ligand PD-L1 inhibitors include Atezolizumab (trade name: Tecentriq) and Avelumab And so on. Meanwhile, Ipilimumab (trademark: Yervoy) and the like have been approved by the FDA as CTLA-4 inhibitors that inhibit the interaction of CTLA-4 / B7-1 / B7-2. In recent years there has been an impressive success, especially in patients with metastatic melanoma or hodgkin lymphoma, and there are many possibilities in clinical trials in other types of cancer patients.
  • a pharmaceutical composition for treating cancer comprising, as an active ingredient, a signal-regulatory protein or a fusion protein comprising the signal-regulating protein and an immunological cell death-inducing agent.
  • the signal regulating protein may be Sirp alpha, Sirp alpha, or a high affinity variant thereof.
  • the signal regulatory protein may be composed of an amino acid sequence selected from the group consisting of SEQ ID NOS: 5, 7, 9, 11, 13 and 15 to 65.
  • the fusion protein may include the signal regulatory protein and the functional peptide, and may further include a linker peptide between the signal regulatory protein and the functional peptide.
  • the functional peptide may be a multimerization domain for massimizing the fusion protein, a protein that specifically binds to a cancer cell-specific receptor or ligand, an immunomodulatory polypeptide comprising a costimulation domain, a chemokine receptor, Lt; / RTI > ligand.
  • the multimerization domain may be a dimerization domain, a trimerization domain, a tetramerization domain, a hexamerization domain, a 12merization domain, or a 24merization domain
  • domain is an Fc fragment, receptor tyrosine kinase (RTK) of the cytoplasmic domain, dimerization domain of kinesin, dimerization domain, Tol- like receptor (TLR) of fibronectin comprising the heavy chain hinge region and CH 2 and CH 3 parts of the antibody
  • TLR Tol- like receptor
  • the trimerization domain is selected from the group consisting of a trimerization domain of collagen, a trimerization domain of TRAIL, a trimerization domain of an Eml4 protein, or a trimerization domain of Cllthrin
  • the tetramerization domain may be a tetramerization domain of p53, a tetramerization domain of DsRed, or
  • cancer cell specific receptor or ligand refers to a cell surface receptor or ligand that is specifically expressed in cancer cells.
  • Such cancer cell specific receptor or ligand includes epithelial growth factor receptor (EGFR), somatostatin receptor (SSTR), ⁇ v ⁇ 5 integrin, vascular endothelial growth factor receptor (VEFGR), human epithelial growth factor receptor 2 (HER2), androgen receptor (AR), estrogen receptor (ER), progesterone receptor (PR) (PD-1L), MUC1, MUC2, MUC3, folate receptors, ErbB2, transferrin receptors, TAG-1 receptors, bombesin receptors, prostate-specific G- 72, G M3 , Le x , CD10, CD20, or CEA.
  • the protein specifically binding to the cancer cell-specific receptor or ligand may include an antibody, a functional fragment thereof, or an antibody analogue that specifically binds to the cancer cell-specific receptor or ligand And may be a protein that specifically binds to the cancer cell-specific receptor or ligand, for example, a protein having an RGD domain that specifically binds to an integrin expressed specifically in a cancer cell.
  • the term " antibody” refers to a Y-shaped protein produced from plasma cells used by the immune system to identify or neutralize exogenous substances, such as bacteria or viruses, also referred to as immunoglobulins.
  • the antibody used in this document includes various " functional fragments ", such as Fab, F (ab ') 2, Fab', ScFv and sdAb, from an antibody.
  • the term "functional fragment of an antibody" used in the present specification includes all of the fragments generated by recombinant methods, as well as fragments obtained by digesting an antibody with a protein cleaving enzyme.
  • Fab is an antigen-binding antibody fragment that is produced by digesting an antibody molecule into a protease, papain, and is a dimer of two peptides of VH-CH1 and VL- , And another fragment generated by papain is referred to as Fc (fragment crystallizable).
  • F (ab ') 2 refers to a fragment comprising an antigen binding site in a fragment produced by digesting an antibody with pepsin, a protease, and the form of a tetramer in which two Fabs are linked by a disulfide bond .
  • Another fragment produced by pepsin is referred to as pFc '.
  • Fab &quot is a molecule similar in structure to Fab produced by the separation of F (ab ') 2 under weakly reducing conditions.
  • ScFv is an abbreviation of " single chain variable fragment ", which is not a fragment of an actual antibody.
  • the heavy chain variable region (VH) and the light chain variable region (VL) (Glockshuber et al., Biochem. 29 (6): 1362-1367, 1990), although it is not a unique antibody fragment as a kind of fusion protein prepared by linking with a linker peptide of the size
  • sdAb single domain antibody
  • sdAb single domain antibody
  • antibody mimetic is intended to mean that, unlike conventional full-length antibodies, in which two heavy chains and two light chains form a quaternary structure of a heterozygous complex, Chain variable fragment (scFv), which is an artificial fragment linked by a linker to a variable region of Fab, F (ab ') 2, Fab' or heavy and light chains,
  • scFv Chain variable fragment
  • a concept comprising an antibody-like protein prepared from a non-antibody-derived protein scaffold such as an antibody fragment (V H H, V NAR, etc.) derived from camel or cartilaginous fish consisting only of heavy chain or nanobody, monobody, variable lymphocyte receptor (VLR) to be.
  • the term " costimulatory domain" refers to a cytoplasmic domain responsible for the T cell-assisted stimulatory function of the costimulatory factor, an immunological protein that assists T / NK activation it means.
  • auxiliary stimulatory domains include CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), BTLA (B and T lymphocyte associated protein), DR3 1BB, CD2, CD40, CD30, CD27, SLAM (signaling lymphocyte activation molecule), 2B4 (CD244), NKG2D / DNAX- activating protein 12, TIM1 immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, lymphocyte activation gene 3, B7-1, B7-H1, glucocorticoid-induced TNFR family related protein, HVEM mediator) or the cytoplasmic domain of OX40L [ligand
  • the immunomodulatory polypeptide is selected from the group consisting of CD28, ICOS, CTLA4, PD1, BTLA, DR3, 4-1BB, CD2, CD40, CD30, CD27, SLAM, 2B4, NKG2D) / DAP12, TIM1 , TIM2, TIM3, TIGIT, CD226, CD160, LAG3, B7-1, B7-H1, GITR, HVEM or OX40L or their complementary stimulatory domains. and Flies, DB, Nat. Rev. Immunol . 13 (4): 227-242, 2013).
  • &quot refers to a chemotactic cytokine that modulates cell migration and location by activating a G protein-linked chemokine receptor (GPCR) that includes a 7-transmembrane portion.
  • GPCR G protein-linked chemokine receptor
  • Chemokines are divided into four subfamilies, CC, CXC, CX3C and XC, depending on the location of the first two N-terminal cysteine residues.
  • a tumor microenvironment composed of cells of the host surrounding the tumor and the tumor
  • tumor-associated host cells and cancer cells secrete various chemokines
  • Various types of cells that mediate the balance between tumor and tumor-promoting responses are replenished and activated.
  • chemokines also participate in other tumor-related processes, including tumor cell growth, neovascularization and metastasis. Therefore, a polynucleotide encoding a chemokine receptor that is not expressed in NK101 of the present invention can be transduced and expressed by increasing the specific mobility of NK101 cells of the present invention against cancer cells (Yang et al ., J. Immunother Cancer , 3 (Suppl 2): P24, 2015).
  • the chemokine receptor may be CCR or CXCR
  • the CCR may be CCR1, CCR2, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10
  • the CXCR may be CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CXCR6, or CXCR7.
  • apoptosis inducing ligand refers to a protein that binds to a receptor on the surface of a cell and induces apoptosis of the target cell.
  • apoptosis inducing ligands include TRAIL (TNF-related apoptosis-inducing ligand) and FasL (Fas ligand).
  • the apoptosis inducing ligand may be TRAIL or FasL.
  • linker peptide is selected from the group consisting of (G 4 S) n, (GSSGGS) n, KESGSVSSEQLAQFRSLD (SEQ ID NO: 5), EGKSSGSGSESKST (SEQ ID NO: 66), GSAGSAAGSGEF (SEQ ID NO: 68), A (EAAAK) 4 ALEA (EAAAK) 4 A (SEQ ID NO: 69), GGGGGGGG (SEQ ID NO: 70), GGGGGG (SEQ ID NO: 71), GGGGS No.
  • the fusion protein may further include a tag peptide for purification at the N-terminal or C-terminal for efficient purification.
  • the tag peptide comprises a HisX6 peptide, a GST peptide, a FLAG peptide (DYKDDDK, SEQ ID NO: 83), a streptavidin binding peptide, a V5 epitope peptide (GKPIPNPLLGLDST, SEQ ID NO: 84), a Myc peptide (EQKLISEE, ), Or HA peptide (YPYDVPDYA, SEQ ID NO: 86).
  • the immunogenic cell death inducer may be selected from the group consisting of an anthracycline anticancer agent, a taxane anticancer agent, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, (GADD34 / PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone or oxaliplatin, and cardiac glycoside may be a non-immunogenic cell
  • the GADD34 / PP1 inhibitor may be used in combination with mitomycin
  • the anthracycline anticancer agent may be selected from the group consisting of daunorubicin, doxorubicin, epirubicin, ), Idarubicin, pixantrone, sabarubicin, or valrubicin, and the taxane family
  • the anti-cancer agent may be paclitaxel or docetaxel, and the anti-EGFR antibody
  • the immunological checkpoint may be PD-1, PD-L1, CTLA-4, B7-1 or B7-2, and the immune checkpoint inhibitor May be a PD-1 / PD-L1 interaction inhibitor or a CTLA-4 / B7-1 / B7-2 interaction inhibitor.
  • the PD-1 / PDL1 interaction inhibitor may be an antibody targeting PD-1 or PDL1 or a functional fragment of the antibody or a single chain-based antibody analog
  • the CTLA- 4 / B7-1 / B7-2 interaction inhibitor may also be an antibody targeting the CTLA-4, B7-1 or B7-2, or a functional fragment of the antibody or a single chain-based antibody analog
  • the PD- 1 or PDL1 may be Pembrolizumab, Nivolumab, Atezolizumab or Avelumab, and the antibody that targets CTLA-4 / B7-1 / B7-2
  • the interaction inhibitor may be Ipilimumab.
  • the single-chain-based antibody analog may be a scFv, a sdAb, a diabody, a monobody, a variable lymphocyte receptor (VLR), a nanobody, Or a camelized heavy chain fragment (VHH).
  • VLR variable lymphocyte receptor
  • VHH camelized heavy chain fragment
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
  • the composition comprising a pharmaceutically acceptable carrier may be various oral or parenteral formulations, but is preferably a parenteral formulation.
  • a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.
  • Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, .
  • Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like.
  • excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • non-aqueous solvent and the suspending agent examples include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
  • injectable ester such as ethyl oleate.
  • the suppository base examples include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.
  • the pharmaceutical composition is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, nonaqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories Any one of the formulations can be used.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally.
  • parenterally it can be administered through various routes such as intravenous injection, intranasal inhalation, intramuscular injection, intraperitoneal administration, percutaneous absorption .
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level will vary depending on the species and severity, age, sex, The activity of the compound, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts.
  • the pharmaceutical composition of the present invention may be administered at a dose of 0.1 mg / kg to 1 g / kg, more preferably at a dose of 1 mg / kg to 500 mg / kg. On the other hand, the dose can be appropriately adjusted according to the age, sex and condition of the patient.
  • the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other anti-cancer agents, and may be administered sequentially or simultaneously with other conventional anti-cancer agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.
  • a signal-regulatory protein or a fusion protein comprising the signal-regulating protein and an immunogenic apoptosis-inducing agent in the manufacture of a pharmaceutical composition for the treatment of cancer .
  • a method for the treatment of cancer comprising administering to a subject in need thereof a multimer of a fusion protein comprising a signal-regulatory protein or a multimerization domain and an immunogenicity cell death inducing agent
  • the method comprising administering to the individual a cancer treatment method comprising the steps of:
  • the immunogenic proliferation inducing agent may be such that when the multimer of the fusion protein forms a nanocage, the loading of the immunogenicity cell death inducing agent which can be loaded into the nanocage can be carried out by using a recombinant exosome , And culturing the genetically engineered cells to produce the nano-cage, wherein the separated nanocage is placed in a solvent in which the anthracycline-based anticancer drug is dissolved and stirred.
  • a complex of an anthracycline type anticancer agent is formed in advance with a divalent metal ion (for example, Cu 2+ , Fe 2+ , and Zn 2+ ), and then the above- Ionic-anthracycline-based anticancer drug complex is immersed in the buffer solution.
  • the anticancer agent can be loaded on the inner pore through the disassemble-reassembly process of the ferritin heavy chain nanocage due to the pH difference, and the anticancer agent can be loaded on the protein nanocage by the pore opening due to the difference in ion concentration .
  • neoantigens which are targets of cancer immunotherapy
  • therapies that increase the antitumor T cell response and, although the mutagenic nature of the cancer cells is very high, Only 1% of expressed mutated proteins cause an immune response in cancer patients.
  • MHC major histocompatibility complex
  • APCs immunogenic neoantigen to the host T cell in the tumor
  • the present inventors have developed a new strategy to overcome the activation-energy limit of immunosuppressive tumor microenvironment and to mediate the delivery and presentation of tumor nodal antigens by APC to host T cells.
  • the strategy is based on naturally derived ferritin-based nanocapsules that include drugs that induce immunogenic cancer cell death (ICD) as well as deliver ligands that enhance cancer cell phagocytosis by APC.
  • ICD immunogenic cancer cell death
  • an anticancer compound nano cage in which doxorubicin is loaded inside a nanocage produced by self-assembly of a ferritin heavy chain protein and a fusion protein composed of Sirp alpha or a specific type of immunocyte such as T lymphocyte in the anticancer compound nanocage
  • an immune checkpoint inhibitor which means a drug of the type that blocks a specific protein produced by some cancer cells, thereby inducing immunogenic cell death of cancer cells to promote tumor antigen-specific tumor immunity
  • developed a next-generation anti-cancer drug that does not have side effects caused by conventional anticancer drugs and can sustain the anti-cancer effect by immune cells even after treatment.
  • FIG. 1A is a schematic diagram showing a schematic view of an anticancer compound nanocage in which doxorubicin is loaded inside a nanocage produced by self-assembly of a ferritin heavy chain protein and a fusion protein composed of Sirp alpha according to an embodiment of the present invention.
  • a fusion protein in which a Sirp alpha or Sirp gamma protein is linked to the C-terminus of a ferritin heavy chain protein is expressed, self-assembly of the ferritin heavy chain protein 24 subunit forms an empty ferritin nanocage.
  • the combined administration of the multispecific Sirp alpha protein and doxorubicin according to an embodiment of the present invention not only infiltrates macrophages, dendritic cells and CD8 + cells, which are innate immune cells, into tumor tissue (FIGS. 3A, 3B, 5), activating immune cells in the lymph nodes and spleen around the tumor tissue, and obtaining cancer-specific immunity (FIG. 4).
  • the combination administration of the multispecific Sirp alpha protein and doxorubicin of the present invention has a very effective anticancer activity in CT26.CL25 colorectal cancer cells, CT26 colorectal cancer cells and B16F10-Ova melanoma cells (Figs. 8A to 9B).
  • the conjugate of the present invention delivers drugs that induce immunogenic cancer cell death (ICD) as well as ligands that enhance cancer cell phagocytosis by APCs (antigen presenting cells).
  • ICD immunogenic cancer cell death
  • APCs antigen presenting cells
  • the combination drug of the present invention induces the release of danger signals and neoantigens in dying cancer cells, enhances tumor cell phagocytosis, and induces tumor-specific T cells (DCs) by dendritic cells loaded with a neonatal antigen peptide To cross-priming the immune system of the host against the presence of cancer cells and obtaining an intrinsic anti-cancer vaccination against cancer.
  • DCs tumor-specific T cells
  • Congenital immune cells such as macrophages and dendritic cells mediate the activity of the adaptive immune system through phagocytosis and antigen presentation and play an important role in initial host defense against pathogens
  • One mechanism to avoid phagocytosis by innate immune cells is to "up-regulate CD47, the Do not eat me” signal, to block the CD47-Sirp ⁇ axis between tumor cells and phagocytic cells
  • CD47-based therapies have been shown to increase the number of congenital and adaptive immune responses in immunocompetent mouse models, (Liu, XJ et al., Nat Med. 21, 1209-12). 15, 2015.)
  • Sirp ⁇ mutants and nano-bodies that block CD47 as a cancer treatment have been developed, but the anticancer activities of these Sirp ⁇ mutants themselves are not considered to be superior to what they seem to be.
  • the present inventors have found that, as a result of intensive efforts, the present inventors have found that a Sirp protein capable of binding and antagonizing human and mouse CD47 can be fused with human ferritin heavy chain protein
  • a multimerized fusion protein and an immunogenicity cell death inducer such as doxorubicin
  • the function of these Sirp proteins is remarkably improved to inhibit the immune cell evasion of cancer cells and to prevent the death of cancer cells by immune cells It can be confirmed that it can be promoted.
  • the present inventors have found that the combined treatment of the multispecific Sirp and the immunogenicity cell death inducer stimulates the local inflammatory reaction, And more strongly induces the production of dendritic cells. Some dying cancer cells trigger a massive immune response, which is called 'immunogenic cell death' (Kroemer et al ., Annu. Rev. Immunol. 31: 51-72, 2013).
  • the ICD communicates with a combination of three distinct 'risk' signals, spatially limited.
  • Doxorubicin an anthracycline-based anticancer drug that induces three characteristics of ICD in cancer cells treated with ICD inducers for delivery with multispecific Sirp was selected and the advantages of metal ion-binding affinity of ferritin, Was used.
  • the metal-ion binding affinity of the ferritin allows a metal-based drug or metal-complex drug to accumulate in the central cavity of the ferritin.
  • the present inventors prepared a doxorubicin preparation encapsulated in FHSirp ⁇ nanocage by innoculating doxorubicin pre-complexed with Cu (II) into the interior of the nanocage, and named it FHSirp ⁇ -Dox.
  • Successful loading of doxorubicin into FHSirp ⁇ was confirmed by size-limited chromatography and the amount of doxorubicin captured was found to be 54 doxorubicin molecules per FHSirp ⁇ nanocage.
  • the FHSirpa-Dox was administered to a tumor model mouse, and it was confirmed that there was a strong ascending antitumor activity reflecting a large amount of a mass-multiplied CD47 antagonist and favorable tumor accumulation of ICD.
  • Phagocytosis and maturation of innate immune cells in the immune suppressive tumor microenvironment stimulate local inflammatory responses, leading to effective delivery and presentation of immunogenic tumor neoplasia to T cells.
  • This strong immune response has resulted in total tumor eradication and a persistent anti-tumor immune response and, overall, has proven to be a universal and effective approach to activating the immune system of the host to tumors.
  • Chimeric antigen receptor (CAR) T cell therapy is also associated with other major obstacles including the need for constant expression of the desired target tumor nodal antigen and economic requirements and the cost of in vitro manipulation, and is associated with PD-1 antibodies
  • Such regulatory antibodies have the disadvantage that all active or depleted T cells, including anti-tumor and anti-self-autoimmune T cells, can be stimulated.
  • the combined administration of the multispecific Sirp protein of the present invention and the immunogenicity cell death inducing agent activates both local and systemic anti-tumor specific immunity, and thus the effect thereof as an 'intrinsic anti-cancer vaccination' ,
  • the synergistic effect has a wide potential and can be used for various types of cancer therapy regardless of stage, considering a durable and robust response.
  • a polynucleotide (SEQ ID NO: 4) encoding a linker peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 and a polynucleotide (SEQ ID NO: 6) encoding a Sirp alpha highly compatible variant consisting of the amino acid sequence shown in SEQ ID NO: 5 After cloning by PCR or synthesis, they were ligated using restriction enzymes and ligase, and cloned into an expression vector pT7-7 containing His tag.
  • a restriction enzyme Xho I recognition site was added between hFTH and the linker peptide, and a restriction enzyme Hin d III recognition site was added between the linker peptide and Sirp ⁇ , And a restriction enzyme Cla I recognition site was added to the 3'-end of the polynucleotide encoding Sirp alpha.
  • the vectors prepared above were transformed into E. coli by the method described by Hanahan (Hanahan D, DNA Cloning vol. 1, 109-135, IRS press 1985). Specifically, the above-prepared vectors were transformed with Escherichia coli BL21 (DE3) treated with CaCl 2 by heat shock method, cultured in a medium containing ampicillin, and the expression vector was transformed to select cells showing resistance to ampicillin Respectively. The transformed cells were cultured at 36 ° C. until the OD 600 reached 0.6, and the expression of the fusion protein was induced by adding 1 mM IPTG and further cultured at 20 ° C. for 16 hours.
  • the cultured cells were collected, disrupted by sonication, and centrifuged at 12,000 g for 30 minutes to remove cellular debris.
  • the recombinant proteins were each separated using a Ni 2+ -NTA column (Qiagen, Hilden, Germany) (wash buffer: pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 80 mM imidazole; elution buffer: mM sodium phosphate, 300 mM NaCl, 250 mM imidazole).
  • the buffer was replaced with PBS using a membrane filter (Amicon, 10K) to remove imidazole from the elution buffer.
  • the concentration of the obtained nanocage was measured by Bradford protein analysis method.
  • the nanocage thus prepared was named 'FHSirp ⁇ HV'.
  • the inventors of the present invention carried out the same method as in Example 1-1 except that the polynucleotide encoding the human Sirp alpha wild type protein (SEQ ID NO: 8) described in SEQ ID NO: 7, which is not a Sirp alpha high affinity mutant, A fusion protein to which a Sirp? Wild-type protein is linked and a ferritin heavy chain nano cage using the same (hereinafter, abbreviated as 'FH-hSirp? WT nanocage').
  • the present inventors prepared a Sirp gamma wild type (SEQ ID NO: 10) in the same manner as in Example 1-1 except that the polynucleotide encoding the Sirp gamma wild type protein (SEQ ID NO: 10), which is not the Sirp alpha high affinity mutant, (Hereinafter abbreviated as " FHSirp gamma WT nanocage ") in which a protein is linked.
  • a polynucleotide encoding a Sirp gamma mutant (hereinafter abbreviated as " Sirp gamma V1 ") having an amino acid mutation corresponding to the mutated amino acid of the above Sirp alpha high affinity mutant in a Sirp gamma wild type protein which is not a Sirp alpha high affinity mutant (Hereinafter abbreviated as 'FHSirp ⁇ V1 nanocage') in which a Sirp ⁇ V1 protein was linked to a ferritin heavy chain protein was prepared in the same manner as in Example 1-1, except that the ferritin heavy chain protein (SEQ ID NO: 12) .
  • a Sirp gamma mutant having an amino acid mutation corresponding to the mutated amino acid of the above Sirp alpha high affinity mutant (hereinafter referred to as " Sirp gamma mutant ") except for the valine in which the 27th amino acid is not substituted in the Sirp gamma wild type protein, (SEQ ID NO: 14) encoding the Sirp gamma V2 protein was linked to the ferritin heavy chain protein in the same manner as in Example 1-1 except that the polynucleotide encoding the Sirp gamma V2 protein (abbreviated as " HV2 & Hereinafter abbreviated as " FHSirp gamma V2 nanocage ").
  • the present inventors firstly reacted 1 mg / ml of doxorubicin with 1 mM of copper ion (Cu 2+ ) at room temperature for 30 minutes to form a doxorubicin-copper ion complex. Then, the mixture was added to the FHSirp? HV solution (250 ⁇ ⁇ / ml) prepared in Example 1-1 and reacted at room temperature for 120 minutes. The reactants were free of free doxorubicin and copper ions by chromatography on a PD-10 column. The loaded doxorubicin was measured by a fluorescence spectrometer (2103 EnVision TM Multilabel Plate Readers, PerkinElmer, USA) and quantitated in comparison with a standard curve.
  • the particle size of the nanocage was analyzed using a dynamic light scattering (DLS) analyzer (Malvern zetasizer nano ZS, UK), and a nanocage produced by transmission electron microscopy was photographed.
  • DLS dynamic light scattering
  • the doxorubicin composite nano cage of the present invention also showed a single peak on the FPLC, and the absorbance at 480 nm for the doxorubicin measurement also showed the same retention as the protein detection at the wavelength of 280 nm Time, it was indirectly confirmed that doxorubicin was successfully loaded into the doxorubicin composite nanocage of the present invention.
  • the particle size analysis results also showed that the nanoparticles had a particle size of 10 to 100 nm (average 19.38 1.7 nm).
  • the transmission electron microscope photographs also showed spherical nanoparticles Respectively.
  • SEQ ID NO: 90 encoding the Fc domain of human IgG1 described in SEQ ID NO: 89 and a polynucleotide (SEQ ID NO: 4) encoding a linker peptide consisting of the amino acid sequence of SEQ ID NO:
  • the polynucleotide encoding the fusion protein to which the polynucleotide encoding the Sirp alpha high-affinity mutant (SEQ ID NO: 6) consisting of the amino acid sequence represented by SEQ ID NO: 5 is linked is cloned by PCR or synthetic method, And then cloned into an expression vector pT7-7 containing His tag.
  • a restriction enzyme Xho I recognition site was added between Fc and linker peptide, and a restriction enzyme Hin d III recognition site was added between the linker peptide and Sirp alpha , And a restriction enzyme Cla I recognition site was added to the 3'-end of the polynucleotide encoding Sirp alpha.
  • the vectors prepared above were transformed into E. coli by the method described by Hanahan (Hanahan D, DNA Cloning vol. 1, 109-135, IRS press 1985). Specifically, the above-prepared vectors were transformed with Escherichia coli BL21 (DE3) treated with CaCl 2 by heat shock method, cultured in a medium containing ampicillin, and the expression vector was transformed to select cells showing resistance to ampicillin Respectively. The transformed cells were cultured at 36 ° C. until the OD 600 reached 0.6, and the expression of the fusion protein was induced by adding 1 mM IPTG and further cultured at 20 ° C. for 16 hours.
  • the cultured cells were collected, disrupted by sonication, and centrifuged at 12,000 g for 30 minutes to remove cellular debris.
  • the recombinant proteins were each separated using a Ni 2+ -NTA column (Qiagen, Hilden, Germany) (wash buffer: pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 80 mM imidazole; elution buffer: mM sodium phosphate, 300 mM NaCl, 250 mM imidazole).
  • the buffer was replaced with PBS using a membrane filter (Amicon, 10K) to remove imidazole from the elution buffer.
  • the concentration of the obtained fusion protein was measured by Bradford protein analysis method.
  • the fusion protein thus prepared was named 'FcSirp ⁇ '.
  • the inventors of the present invention conducted experiments to confirm whether the combination drug (FHSirp alpha -Dox) of Sirp alpha oligosaccharide and doxorubicin prepared according to an embodiment of the present invention showed the same anti-cancer effect in an animal model experiment,
  • the anticancer activity of FHSirp alpha -Dox prepared in Example 2 of the present invention was examined.
  • Balb / c wild type mouse was used as an experimental animal, and experiments on the above experimental animals were carried out according to the regulations of the KIST animal ethics committee.
  • each substance is 1 mg / kg of doxorubicin (dox), 15 mg / kg of wt FH-dox (equivalent to 1 mg / kg of doxorubicin), 9.5 mg / kg of dox , And FHSirpa-dox 28 mg / kg (corresponding to 1 mg / kg as the doxorubicin content).
  • the length (L) and the width (W) of the cancer cells were measured using a caliper at intervals of 3 days until the 25th day after the injection of the cancer cells, and then calculated using the following formula (Fig. 2a)
  • the tumor tissue was taken and the tumor tissue was weighed to determine the weight of the tumor tissue ( Figures 2b to 2d):
  • Figs. 2B and 2C the size of the tumor exceeded 1,000 mm 3 in the control group (only the buffer solution was injected), and the effect of doxorubicin alone treatment was also insignificant.
  • the combination of wild-type doxorubicin with doxorubicin (wtFH-dox) and wild-type Sirp ⁇ with doxorubicin (mSirp ⁇ + dox) showed anticancer effects but did not sufficiently inhibit the growth of cancer cells.
  • the FHSirp alpha-dox combination of Sirp alpha oligosaccharide and doxorubicin according to one embodiment of the present invention significantly inhibited the growth of cancer cells to a level of 200 mm 3 or less even after 25 days. Furthermore, in the case of FHSirp ⁇ -dox, the combination of Sirp ⁇ multimer and doxorubicin according to an embodiment of the present invention almost completely inhibited the growth of cancer cells, and thus the cancer cells could not be distinguished visually.
  • FIG. 2d when the tumor cells were sacrificed after 25 days of the injection of the cancer cells, the size of the tumor tissues was measured.
  • the Sirpa multimer of the present invention and doxorubicin were administered concomitantly, (WtFH-dox) with doxorubicin encapsulated in normal ferritin without Sirp ⁇ oligos, the combination of doxorubicin with monomeric recombinant Sirp ⁇ , and the inhibition of cancer cell growth by doxorubicin alone.
  • the present inventors performed flow cytometry analysis on cancer cells to determine whether immunogenetic cell death is induced by the combined administration of Sirp alpha oligonucleotide and doxorubicin according to one embodiment of the present invention.
  • test substance (buffer, dox, wtFH-dox, mSirp? +?)
  • dox doxorubicin
  • wt FH-dox equivalent to 1 mg / kg of doxorubicin
  • doxorubicin equivalent to 1 mg / kg of doxorubicin
  • doxorubicin equivalent to 1 mg / kg of doxorubicin
  • FHSirpa-dox 28 mg / kg (corresponding to 1 mg / kg as the doxorubicin content).
  • tumor tissues were harvested and monoclonalized with DNase and collagenase, and tumor-microenvironmental dendritic cells and macrophages were treated with anti-CD11c antibody and anti-F4 / 80 antibody (FACS analysis) (Figs. 3A and 3B).
  • CT26 of the mouse spleen and tumor-draining lymph node by FHSirp alpha-dox was administered with FHSirp alpha oligonucleotide and doxorubicin.
  • FHSirp alpha oligonucleotide and doxorubicin were administered with FHSirp alpha oligonucleotide and doxorubicin.
  • Cellular immune responses to CL25 cancer cells were investigated. That is, the level of interferon-gamma (INF- ⁇ ) of T cells specific to ⁇ -galactosidase, an antigen of CT26.CL25, was quantified by ELISA (R & D Systems, Inc, USA) analysis.
  • mice per group Three mice per group were selected from the mice treated in the same manner as in Experimental Example 1-1, and the tumor-drained lymph node (TDLN) was extracted from each mouse, and the tissue was cultured in a sterile petri dish And cells were separated from the tissue patches of the splenic and tumor draining lymph nodes using a cell strainer. All the contents in the Petri dish were transferred to a 15 ml tube and filled with RPMI 1640 medium. After centrifugation at 500 G for 10 minutes, red blood cells were hemolyzed by using a red blood cell lysis buffer (Sigma-Aldrich, Germany) to the pellet from which the supernatant was removed .
  • red blood cell lysis buffer Sigma-Aldrich, Germany
  • TDLN tumor-drained lymph node
  • splenocytes ⁇ -gal peptide
  • TPHPARIGL ⁇ -gal peptide
  • SEQ ID NO: 87 Including a naturally engineered H-2 Ld restriction epitope including the naturally occurring H-2 Ld restriction epitope, including the CTL determinant derived from the gp70 protein epitope AH1 (SPSYVYHQF, SEQ ID NO: 88, CT26), which is an endogenous antigen of CT26.CL25
  • P1A peptide negative control
  • the FHSirp ⁇ nano cage loaded with doxorubicin according to one embodiment of the present invention recorded a level of interferon gamma expression in the splenocytes of 100 pg / ml or more, indicating that the immune activation degree of cancer cells was very high.
  • mice In order to confirm whether the results of Experimental Examples 1-3 were effective for the immune cell aggregation into tumor cells, the present inventors flipped the tumor tissues extracted in Experimental Example 1-3 to prepare T cell marker CD8 Immunohistochemical analysis was performed on the mice.
  • the tumor tissues obtained from the FHSirp ⁇ -dox-treated experimental animals prepared in Experimental Example 1-3 were fixed in a 10% neutral formalin solution to prepare paraffin blocks, which were prepared into 4 ⁇ m thick flakes, After washing three times with TBST (Tris-buffered saline, Tween-20), the cells were incubated with Renaissance Ab diluent (PD905, Biocare Medical) for blocking. (MP-7445-15, Vector Laboratories, USA) and incubated for 3 min at room temperature for 10 min.
  • TBST Tris-buffered saline, Tween-20
  • TSA triamine signal amplification
  • CD8 + T cells were stained positive in tumor tissues. This demonstrates that CD8 + T cells infiltrate into tumor tissues, suggesting that the anticancer effect is due to the synergistic action of immune cell replacement following CD47 masking of doxorubicin and Sirpa.
  • the inventors of the present invention investigated whether the combination of Sirpa multimer and doxorubicin (FHSirp? -Dox) according to one embodiment of the present invention exhibits an equivalent anticancer effect in intratumoral injection as well as intravenous injection.
  • cancer cells were induced by subcutaneous inoculation of 1 x 10 6 cells of CT26.CL25 cancer cells in an 8-week-old Balb / c wild-type mouse on the left and the like.
  • each test substance buffer, wtFH-dox and FHSirp ⁇ -dox
  • the dose of each substance corresponds to wt FH-dox 15 mg / kg (corresponding to 1 mg / kg for doxorubicin content) and FHSirp ⁇ -dox 28 mg / kg (for doxorubicin 1 mg / kg ).
  • the volume of cancer cells was measured at intervals of 3 days until the 25th day after the injection of the cancer cells as described in Experimental Example 1-1, and the tumor tissues of the experimental animals were removed at 25 days and the tumor tissues were weighed.
  • Nano cage according to one embodiment of the present invention is effected by recruiting immune cells Assuming that the tumor tissue after FHSirp-dox administration according to one embodiment of the present invention was transplanted to the other side of the same animal not sacrificed, the growth of the cancer at the transplanted position was analyzed over time, The number of surviving mice was counted.
  • FIGS. 7A and 7B in the animal to which the tumor tissue harvested from the FHSirp alpha-dox administration group according to the embodiment of the present invention was transplanted, only one mouse in which the cancer cells were grown until 28 days passed And mice transplanted with tumor tissues extracted from the group of administration of other substances showed cancer recurrence although there was a degree of difference with the lapse of time.
  • FIG. 7C in the FHSirp ⁇ -dox administration group according to one embodiment of the present invention, no surviving experimental animals were observed until 80 days after the passage of 80 days. In the case of FHSirp ⁇ , survival rate after 80 days was 80%, which was better after FHSirp ⁇ -Dox, and survival rate after 80 days after doxorubicin alone or recombinant Sirp ⁇ alone was 50%.
  • the nanocage according to an embodiment of the present invention not only inhibits the growth of cancer cells but also provides a memory effect on immune cells capable of inhibiting recurrence after cancer treatment. Therefore, the nanocage according to one embodiment of the present invention can be very effective not only for treatment of cancer but also for suppression of recurrence.
  • Balb / c wild-type mice were subcutaneously inoculated with 1 ⁇ 10 6 cells of CT26 cancer cells on the left side and cancer cells were induced on the 7th day after the injection of the cancer cells.
  • Each test substance (buffer, dox, wtFH-dox, mSirp ⁇ + dox , FHSirp ⁇ -dox) were injected intravenously 5 times at intervals of 3 days.
  • the dose of each substance is 1 mg / kg of doxorubicin (dox), 15 mg / kg of wt FH-dox (equivalent to 1 mg / kg of doxorubicin), 9.5 mg / kg of dox , And FHSirpa-dox 28 mg / kg (corresponding to 1 mg / kg as the doxorubicin content).
  • dox doxorubicin
  • FH-dox equivalent to 1 mg / kg of doxorubicin
  • 9.5 mg / kg of dox 9.5 mg / kg of dox
  • FHSirpa-dox 28 mg / kg (corresponding to 1 mg / kg as the doxorubicin content).
  • the present inventors analyzed the anticancer activity against B16F10-Ova cells, a kind of melanoma expressing ovalbumin.
  • the inventors of the present invention specifically injected 1 ⁇ 10 6 cells of B16F10-Ova cancer cells into 8-week-old Balb / c wild type mice by subcutaneously inoculating them on the left side and induced cancer.
  • wtFH-dox, mSirp ⁇ + dox, and FHSirp ⁇ -dox were injected intravenously three times at intervals of three days.
  • the dose of each substance is 1 mg / kg of doxorubicin (dox), 15 mg / kg of wt FH-dox (equivalent to 1 mg / kg of doxorubicin), 9.5 mg / kg of dox , And FHSirpa-dox 28 mg / kg (corresponding to 1 mg / kg as the doxorubicin content).
  • dox doxorubicin
  • FH-dox equivalent to 1 mg / kg of doxorubicin
  • 9.5 mg / kg of dox 9.5 mg / kg of dox
  • FHSirpa-dox 28 mg / kg (corresponding to 1 mg / kg as the doxorubicin content).
  • Cross-priming is the process by which a specific antigen-presenting cell ingests and processes extracellular antigens and presents it with CD8 + T cells (cytotoxic T cells) along with MHC type 1 molecules (cross- presentation refers to a phenomenon in which untouched CD8 + T cells are stimulated to differentiate into cytotoxic CD8 + T cells.
  • mice 8-week-old Balb / c wild-type mice were subcutaneously inoculated with 1 ⁇ 10 6 cells of B16F10-Ova cancer cells on the left side, and on the 7th day after inoculation of cancer cells, FHSirp ⁇ -dox 28 mg / kg (1 mg / kg as doxorubicin) was injected intravenously three times in total every 3 days.
  • FHSirp ⁇ -dox 28 mg / kg (1 mg / kg as doxorubicin) was injected intravenously three times in total every 3 days.
  • cancer tissues Two days after the last intravenous injection, cancer tissues were extracted, monoclonalized with DNase and collagenase, and CD11c-positive cells (dendritic cells) were isolated by a magnetic-activated cell sorting (MACS) method. Thereafter, co-cultivation was performed for 3 days together with OT-1 cells as a non-contact CD8 + T cell, and then the culture supernatant was obtained and the amount of I
  • INF- ⁇ was secreted by 100 pg / ml or more in the FHSirp ⁇ -dox-treated group according to one embodiment of the present invention, whereas INF- ⁇ expression was extremely small in the control group.
  • the dendritic cells of the FHSirp? -Dox-administered group according to an embodiment of the present invention have a cross-priming ability to stimulate noncontact CD8 + T cells by presenting cancer cell antigens on MHC 1 type molecules and non-contact CD8 + T cells .
  • mice 816-week-old Balb / c wild-type mice were subcutaneously inoculated with 1 ⁇ 10 6 cells of B16F10-Ova cancer cells on the left side and cancer cells were induced on the 7th day after inoculation with each test substance (buffer, dox, FHSirp ⁇ and FHSirp ⁇ -dox ) Were injected intravenously twice at intervals of 3 days.
  • the dose of each test substance used was 1 mg / kg for doxorubicin (dox), 28 mg / kg for FHSirp ⁇ , and 28 mg / kg (1 mg / kg for dox) of FHSirpP ⁇ -dox.
  • OT-1 T cells stained with CFSE (carbolxyfluorescein succinimidyl ester) were injected intravenously, and after 3 days, the tumor draining lymph node (TDLN) After celling, the cross-priming ability of each group was analyzed by confirming the degree of cell proliferation of OT-1 T cells through CFSE proliferation analysis by FACS analysis.
  • the present inventors investigated the anticancer effect of the combined use of the FcSirpa fusion protein prepared in Example 3 and the anticancer agent mitoxantrone in order to confirm whether the combined effect of Sirp alpha with the anticancer agent is realized on other platforms.
  • BMDM bone marrow-derived macrophages
  • the HT29 cancer cell line stained with RPMI SE (Thermo Fisher Scientific, USA) with 120 ng / ml of bone marrow-derived macrophages prepared in RPMI medium was incubated at 37 ° C for 2 hours with the control or FcSirp ⁇ 5 < / RTI > or 400 nM of FHSirp alpha prepared in Example 1.
  • FcSirp ⁇ 5 ⁇ M and FHSirp ⁇ 400 nM have the same amount of Sirp ⁇ .
  • the degree of cancer cell phagocytosis was analyzed using a fluorescence microscope (Fig.
  • FIGS. 12A and 12B red: cancer cell proliferated, green: bone marrow-derived macrophage) and quantified (Fig. As a result, as shown in FIGS. 12A and 12B, FcSirp ⁇ and FHSirp ⁇ significantly increased cancer cell phagocytic ability of macrophages, and FcSirp ⁇ showed the best phagocytic capacity.
  • the present inventors then analyzed the phagocytosis of cancer cells using a flow cytometer with different concentrations of FcSirp ⁇ in order to determine the concentration of FcSirp ⁇ phagocytic activity.
  • BMDM was stained with 1 ⁇ M of CellTracker Green CMFDA (Thermo Fisher Scientific, USA). Stained macrophages of 20x10 4 cells were each inoculated into a 35 mm Peptri dish with 2 ml of RPMI medium.
  • the present inventors investigated the synergistic effect of FcSirp ⁇ and mitoxantrone, which is an immunogenic cell death inducer, in combination.
  • MTX mitoxantrone
  • FcSirp ⁇ + MTX group mitoxantrone was administered by intra-tumor injection at a dose of 400 ⁇ g FcSirp ⁇ 4 h after 10 ⁇ g tumor injection.
  • the cancer size was measured at intervals of 3 days (Fig. 13A), and the survival rate up to 21 days after the injection of cancer cells was recorded (Fig. 13B). On the 21st day after the injection of cancer cells, (Fig. 13C).
  • Figs. 13a to 13c the MTX + FcSirp ⁇ group showed the best survival rate and anticancer effect.
  • the results of the experiments show that the immune response of the individual, especially the innate immune response, is enhanced by the combined administration of a signal regulatory protein such as Sirp alpha and an immunogenic apoptosis inducing agent such as doxorubicin or mitoxantrone, These anticancer activities show that Sirp's mass-multiplication plays a very important role.
  • a signal regulatory protein such as Sirp alpha
  • an immunogenic apoptosis inducing agent such as doxorubicin or mitoxantrone
  • the pharmaceutical composition according to one embodiment of the present invention can be used for the production of a more effective anticancer agent.
  • SEQ ID NO: 1 is the amino acid sequence of the human ferritin heavy chain protein and SEQ ID NO: 2 is the nucleic acid sequence of the polynucleotide encoding the human ferritin heavy chain protein.
  • SEQ ID NO: 3 is the amino acid sequence of the linker peptide and SEQ ID NO: 4 is the nucleic acid sequence of the polynucleotide encoding the linker peptide.
  • SEQ ID NO: 5 is the amino acid sequence of the Sirp alpha high affinity mutant protein and SEQ ID NO: 6 is the nucleic acid sequence of the polynucleotide encoding the Sirp alpha high affinity mutant protein.
  • SEQ ID NO: 7 is the amino acid sequence of the human wild-type Sirp alpha protein and SEQ ID NO: 8 is the nucleic acid sequence of the polynucleotide encoding the human wild-type Sirp alpha protein.
  • SEQ ID NO: 9 is the amino acid sequence of the wild-type Sirp gamma protein and SEQ ID NO: 10 is the nucleic acid sequence of the polynucleotide encoding the wild-type Sirp gamma protein.
  • SEQ ID NO: 11 is the amino acid sequence of the Sirp gamma mutant protein 1 (V1)
  • SEQ ID NO: 12 is the nucleic acid sequence of the polynucleotide encoding the Sirp gamma mutant protein 1.
  • SEQ ID NO: 13 is the amino acid sequence of the Sirp gamma mutant protein 2 (V2)
  • SEQ ID NO: 14 is the nucleic acid sequence of the polynucleotide encoding the Sirp gamma mutant protein 2.
  • SEQ ID NOS: 15-65 are amino acid sequences of various Sirpa mutant proteins.
  • SEQ ID NOS: 66-81 are amino acid sequences of various linker peptides.
  • SEQ ID NOS: 82-86 are amino acid sequences of various tag peptides.
  • SEQ ID NO: 87 is the amino acid sequence of? -Gal peptide.
  • SEQ ID NO: 88 is the amino acid sequence of the AH1 peptide.
  • SEQ ID NO: 89 is the amino acid sequence of the IgG1 Fc domain and SEQ ID NO: 90 is the nucleic acid sequence of the polynucleotide encoding the IgG1 Fc domain.

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Abstract

La présente invention concerne une composition pharmaceutique pour le traitement du cancer. La présente invention concerne une composition pharmaceutique pour le traitement du cancer, comprenant comme principes actifs : des protéines régulatrices des signaux ou des protéines de fusion comprenant les protéines régulatrices des signaux ; des multimères des protéines de fusion comprenant des domaines de multimérisation ; et un inducteur de mort cellulaire immunogène.
PCT/KR2018/011505 2017-09-28 2018-09-28 Nouvelle composition pour le traitement du cancer Ceased WO2019066536A1 (fr)

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US20150329616A1 (en) * 2012-12-17 2015-11-19 Trillium Therapeutics Inc. Treatment of CD47+ Disease Cells with SIRP Alpha-FC Fusions
KR20160132117A (ko) * 2014-03-24 2016-11-16 이뮨원코 바이오파마슈티컬즈 (상하이) 컴퍼니 리미티드 새로운 이중 기능성의 재조합 융합 단백질, 이의 제조 방법 및 용도
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KR20160132117A (ko) * 2014-03-24 2016-11-16 이뮨원코 바이오파마슈티컬즈 (상하이) 컴퍼니 리미티드 새로운 이중 기능성의 재조합 융합 단백질, 이의 제조 방법 및 용도
KR20170036796A (ko) * 2014-08-15 2017-04-03 메르크 파텐트 게엠베하 Sirp-알파 면역글로불린 융합 단백질
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