WO2019066536A1 - Composition for treating new cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating cancer. The present invention provides a pharmaceutical composition for treating cancer, comprising as active ingredients: signal-regulatory proteins or fusion proteins including the signal-regulatory proteins; multimers of the fusion proteins including multimerization domains; and an immunogenic cell death inducer.

Description

Composition for treating new cancer

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. In particular, 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. , 13 (1): 54-61, 2007). In the meantime, the above-mentioned cancer treatment strategy through the enhancement of immune function has been attracting attention recently. The correlation study between immune cells and cancer has been carried out in the world since the 1970s, and the immune cells (Pembrolizumab, Merck), which is an anti-cancer immunotherapy antibody, has been accredited approval by the US FDA in September 2014, and has been actively promoting the anti-cancer immunotherapy The importance of research is emerging. In particular, since the immune cells patrol the cancer cells and move around and move, the effect is not limited to the locality but can be used for various cancer cells as well as for monitoring and inhibiting the oncogenesis of the whole body. Therefore, it is necessary to present a new paradigm of cancer prevention and treatment strategies to overcome the cancer microenvironment by controlling the dynamic networking of immune cells in the human body, away from the conventional approach of concentrating on existing cancer cell necrosis.

Disclosure of the Invention 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. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided 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.

According to another aspect of the present invention, there is provided a use of 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 .

According to one aspect of the present invention there is provided 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.

According to one embodiment of the present invention as described above, 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).

Figure 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 < + &gt; 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-γ upon treatment of peptides (β-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 (FHSirpα-dox) according to one embodiment of the present invention, As a result of the chemical analysis, the upper part is a fluorescence staining image of a tumor tissue section after the buffer solution administration, and 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, And the second column on the left shows the result of staining with anti-CD8 antibody. On the left side, the third column shows the result of the DAPI staining and the anti-CD8 antibody staining result And 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, mSirpα + dox and FHSirpα-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 FHSirpα-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, and 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, mSirpα + dox and FHSirpα-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 &Lt; 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, mSirpα + dox and FHSirpirpα-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, FHSirpα, and FHSirpα-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, and 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 FHSirpα and FcSirpα on cancer cells (HT29) according to an embodiment of the present invention, and 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 μM) (***: P < 0.001). &Lt; / RTI &gt;

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.

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).

Definition of Terms

As used herein, 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. Unlike general apoptosis, 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 &quot;. 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.

As used herein, the term " 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. As described above, proteins that are mass-produced by self-assembly are referred to as " self-assembled proteins &quot;, 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.

As used herein, the term " Sirp (signal-regulated protein) " is a regulatory membrane glycoprotein that is expressed predominantly in bone marrow cells and expressed in stem cells or neurons. Sirp &lt; / RTI &gt; and Sirp gamma in the Sirp act as inhibitory receptors and interact with the broadly expressed transmembrane protein, CD47 protein, which is often referred to as the " do not eat me &quot; These interactions negatively regulate the effector function of 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 Sirpα increase the phagocytosis of cancer cells by masking CD47 on cancer cells (Weiskopf et al ., Science 341 (6141): 88-91, 2013).

As used herein, the term " ferritin heavy chain protein &quot; (hereinafter abbreviated as FH) 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.

As used herein, the term " 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. , Sabarubicin, valrubicin, and the like. The mechanism of action of 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 . 9 (13-14): 1530-1539, 2009), dendritic cells 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).

The term " taxanoid anticancer agent " or &quot; taxane anticancer drug &quot; 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. Currently, paclitaxel and docetaxel are presently available. Among them, paclitaxel is a taxane-based chemotherapeutic agent extracted from juniper of Taxus brevifolia . It was approved by the US FDA in 1992 for the treatment of intractable ovarian cancer, 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. Recently, it has been found that 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.

As used herein, the term " 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.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect of the present invention, there is provided 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.

In the pharmaceutical composition for treating cancer, the signal regulating protein may be Sirp alpha, Sirp alpha, or a high affinity variant thereof.

In the pharmaceutical composition for treating cancer, 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.

In the pharmaceutical composition for treating cancer, 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 &gt; ligand.

In the pharmaceutical composition for treating cancer, 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 Or a dimerization domain of tubulin, wherein 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 And the tetramerization domain may be a tetramerization domain of p53, a tetramerization domain of DsRed, or a tetramerization degree of acetylcholine esterase (AChE) May ynyl, the 6-mer localization domain may be a 6-mer of the HSP100 protein localization domain, and the 12-mer localization domain may be a SPD (surfactant protein D, WO2013115608A1) or M. tuberoculosis Hsp16.3, the 24-mer Chemistry The domain may be a ferritin heavy chain protein, a ferritin light chain protein, M. jannanschii Hsp16.5, or yeast Hsp26.

As used herein, the term " 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), α _v β 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.

In the pharmaceutical composition for treating cancer, 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.

As used herein, 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.

As used herein, the term " 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).

As used herein, the term " 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 '.

The term " Fab &quot;, as used herein, is a molecule similar in structure to Fab produced by the separation of F (ab ') 2 under weakly reducing conditions.

As used herein, the term " 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

The term " sdAb (single domain antibody) ", as used herein, refers to an antibody fragment consisting of a single variable region fragment of an antibody, referred to as a nanobody. The sdAb derived mainly from the heavy chain is used, but a single variable region fragment derived from the light chain has also been reported to be a specific binding to the antigen.

As used herein, the term " 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, 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.

As used herein, 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. These 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 for CD134 (OX40), CD252], or a linker of two or more of these.

Wherein 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).

As used herein, the term &quot; chemokine &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. Chemokines are divided into four subfamilies, CC, CXC, CX3C and XC, depending on the location of the first two N-terminal cysteine residues. In particular, in a tumor microenvironment (hereinafter referred to as 'TME') 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. Furthermore, besides the role of chemotaxis, 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).

In the pharmaceutical composition for treating cancer, the chemokine receptor may be CCR or CXCR, and the CCR may be CCR1, CCR2, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10 And the CXCR may be CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CXCR6, or CXCR7.

As used herein, the term " 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. Representative examples of such apoptosis inducing ligands include TRAIL (TNF-related apoptosis-inducing ligand) and FasL (Fas ligand).

In the pharmaceutical composition for treating cancer, the apoptosis inducing ligand may be TRAIL or FasL.

Wherein the 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. 73), PAPAP (SEQ ID NO: 74), (Ala-Pro) n, VSQTSKLTRAETVFPDV (SEQ ID NO: 75), PLGLWA (SEQ ID NO: 76), TRHRQPRGWE (SEQ ID NO: 77), AGNRVRRSVG (SEQ ID NO: 78), RRRRRRRR 79), GFLG (SEQ ID NO: 80), or GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 81).

In addition, in the pharmaceutical composition, 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).

In the pharmaceutical composition for treating cancer, 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, and 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 may be cetuximab.

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.

In the pharmaceutical composition for treating cancer, 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, and 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, wherein 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.

In the pharmaceutical composition for the treatment of cancer, 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).

In the pharmaceutical composition of the present invention, 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. In the case of 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, . In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various 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. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base 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. When administered parenterally, it can be administered through various routes such as intravenous injection, intranasal inhalation, intramuscular injection, intraperitoneal administration, percutaneous absorption .

The composition of the present invention is administered in a pharmaceutically effective amount.

The term " pharmaceutically effective amount " as used herein 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.

According to another aspect of the present invention, there is provided a use of 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 .

According to another aspect of the present invention, there is provided 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. Preferably, 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. In addition, 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 .

Recently, the identification of mutated proteins in tumors known as neoantigens, which are targets of cancer immunotherapy, has led to the development of 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. In order for the new antigen to be immunogenic, it has to be presented after being treated by a major histocompatibility complex (MHC) molecule on the cell surface, and it is activated to efficiently deliver the immunogenic neoantigen to the host T cell in the tumor, (APCs) are loaded only by the loaded neonatal peptide. 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. Accordingly, the inventors of the present invention have found that 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 And 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 And 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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in detail with reference to the accompanying drawings. For ease of explanation, the components may be exaggerated or reduced in size in the drawings.

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. As shown in FIG. 1A, when 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. When such a nanocage is treated with a complex of a metal particle such as copper (Cu) and a drug, the drug can be loaded into the nanocage. The fluorescence FPLC analysis of the FHSirpα nanocage with the generated doxorubicin showed that the doxorubicin was properly loaded inside the nanocage (see FIG. 1b), and the dynamic light scattering analysis and electron microscopy showed that spherical Nanoparticles were formed (see Figs. 1C and 1D)

As a result of the in vivo anticancer activity analysis of the animal model animal with the multimeric FHSirp alpha loaded with the doxorubicin prepared as described above, it was confirmed that the intravenous administration and the intratumoral administration markedly inhibited the growth of cancer cells (Figs. 2c, Figs. 6A to 6C).

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).

In addition, the rechallenge experiment of re-vaccinating the same cancer cells after extracting the tumor tissues in an experimental animal administered with the combination agent of the multispecific Sirp alpha protein of the present invention and doxorubicin showed that the cancer recurrence was suppressed very efficiently without additional administration (See Figs. 7A to 7C).

In addition, the combination administration of the multispecific Sirp alpha protein and doxorubicin of the present invention according to an embodiment 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).

In particular, regarding the mechanism of the anticancer activity against the B16F10-Ova melanoma cells, it was confirmed that the combination administration of the multispecific Sirp alpha protein and doxorubicin of the present invention had cross-priming ability. As a result, by presenting the antigen to CD8 ⁺ T cells was found to accelerate the differentiation and stimulation of the contactless CD8 ⁺ T cells (Figs. 10 to 11b).

The above results indicate that the combined administration of the multispecific Sirp protein and the immunogenic apoptosis inducing agent according to an embodiment of the present invention is effective for the single administration of the Sirp protein and the anti- Suggesting that it may exhibit a very surprising synergistic effect on activity.

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). 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.

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-Sirpα axis between tumor cells and phagocytic cells In addition, 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.) Recently, Sirpα mutants and nano-bodies that block CD47 as a cancer treatment have been developed, but the anticancer activities of these Sirpα mutants themselves are not considered to be superior to what they seem to be.

Thus, in order to improve the CD47-mediated immunotherapy, 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 On the other hand, when such a multimerized fusion protein and an immunogenicity cell death inducer such as doxorubicin are used in combination, 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.

As a result of intensive studies on the strategy for secretion of immunogenic progenitor antigen and risk signal in dying cancer cells, 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.

1) an "eat-me" signal associated with translocation of calreticulin (CRT) to the cell surface present in the ER; 2) a " find-me " signal associated with the activation of ATP secretion; And 3) a signal that promotes the antigenic treatment and presentation of T cells to the extracellular secretion of the nuclear high-mobility group box 1 (HMGB1) protein. These signals regulate a series of receptors expressed on the surface of dendritic cells to stimulate the presentation of tumor nodal antigens to T cells. Therefore, the present inventors have hypothesized that the ICD inducer delivered with the CD47 antagonist to the tumor microenvironment would trigger a danger signal from a dying cancer cell and initiate a cellular immune response.

Doxorubicin (dox), 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 FHSirpα nanocage by innoculating doxorubicin pre-complexed with Cu (II) into the interior of the nanocage, and named it FHSirpα-Dox. Successful loading of doxorubicin into FHSirpα was confirmed by size-limited chromatography and the amount of doxorubicin captured was found to be 54 doxorubicin molecules per FHSirpα 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.

In comparison, current cancer vaccines are limited in that they require constant expression of the desired target tumor nodal antigen and induce the formation of resistant clones that do not express the target antigen. 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. However, 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.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that the invention is not limited to the disclosed embodiments and examples, but may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, It is provided to fully inform the owner of the scope of the invention.

Example 1: Preparation of ferritin nanocage

1-1: Fusion protein of ferritin heavy chain protein and Sirp-α high affinity mutant

The present inventors have found that a polynucleotide encoding a human ferritin heavy chain protein (hFTH) consisting of the amino acid sequence shown in SEQ ID NO: 1 (SEQ ID NO: 2); 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. To facilitate cloning of the polynucleotides encoding the respective proteins or peptides, 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 Sirpα , 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 'FHSirpα HV'.

1-2: Ferritin heavy chain protein and Sirp alpha wild-type fusion protein

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').

1-3: Ferritin heavy chain protein and Sirp gamma wild type fusion protein

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 &quot; FHSirp gamma WT nanocage &quot;) in which a protein is linked.

1-4: Ferritin heavy chain protein and Sirp-gamma mutant 1 fusion protein

The present inventors have found that a polynucleotide encoding a Sirp gamma mutant (hereinafter abbreviated as &quot; Sirp gamma V1 &quot;) 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 'FHSirpγ V1 nanocage') in which a Sirpγ 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) .

1-5: Ferritin heavy chain protein and Sirp-gamma mutant 2 fusion protein

The present inventors have found that 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 &quot; Sirp gamma mutant &quot;) 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 &quot; HV2 & Hereinafter abbreviated as &quot; FHSirp gamma V2 nanocage &quot;).

Example 2: Preparation of doxorubicin loaded 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 占 퐂 / 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 ™ Multilabel Plate Readers, PerkinElmer, USA) and quantitated in comparison with a standard curve. To confirm whether or not the prepared doxorubicin composite nanocage was formed with nanoparticles, 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.

As a result, as shown in FIG. 1B, 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. As shown in FIG. 1C, 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.

Example 3: Preparation of FcSirp alpha

(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. To facilitate cloning of the polynucleotides encoding the respective proteins or peptides, 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 'FcSirpα'.

Experimental Example 1: Preparation of FHSirp? -Dox in vivo  Analysis of anti-cancer effect

1-1: CT26.CL25 Anticancer effect analysis using monoclonal tumor mouse

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.

First, in order to measure the in vivo antitumor activity of various forms of Sirpα protein and doxorubicin, 1 × 10 ⁶ cells of CT26.CL25 cancer cells expressing β-galactose in 8-week-old Balb / c wild-type mice were injected subcutaneously on the left side to induce cancer , 7 days after the injection of the cancer cells, each test substance (buffer, dox, wtFH-dox, mSirpα + dox, FHSirpα-dox) was 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). Then, 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):

Cancer cell volume (V [mm ³ ]) = (L [mm]) x (W [mm]) ² x 0.5

As a result, as shown in 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. In addition, the combination of wild-type doxorubicin with doxorubicin (wtFH-dox) and wild-type Sirpα with doxorubicin (mSirpα + dox) showed anticancer effects but did not sufficiently inhibit the growth of cancer cells. On the other hand, 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 FHSirpα-dox, the combination of Sirpα 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.

The results were slightly different from those of the in vitro test results. In the case of administration of doxorubicin alone or in combination with monomeric recombinant Sirpα, the recombinant Sirpα alone, which caused phagocytosis of cancer cells in macrophages, Considering the fact that the anticancer effect was not so great in model experiments, it is a remarkable result.

In addition, as shown in 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. As shown in FIG. 2d, when the Sirpa multimer of the present invention and doxorubicin were administered concomitantly, (WtFH-dox) with doxorubicin encapsulated in normal ferritin without Sirpα oligos, the combination of doxorubicin with monomeric recombinant Sirpα, and the inhibition of cancer cell growth by doxorubicin alone.

1-2: Immunogenic cell death analysis

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.

Specifically, the present inventors injected test substance (buffer, dox, wtFH-dox, mSirp? +?) In each group from day 7 after inoculation (day 0) of CT26.CL25 cancer cell 1, ¹⁰⁶ cells into 8- dox, FHSirpα-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). On the 25th day after the injection of cancer cells, 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).

As a result, as shown in FIGS. 3A and 3B, it was confirmed that the number of CD11c-positive cells and F4 / 80-positive cells, which are markers of dendritic cells and macrophages, increased more than twice as compared with the control group. This means that FHSirp? -Dox according to an embodiment of the present invention induces a congenital immune response through mobilization of immune cells against cancer cells.

1-3: Analysis of antigen-induced immune response

In order to confirm whether the above-mentioned immune response was specific to cancer cells, CT26 of the mouse spleen and tumor-draining lymph node by FHSirp alpha-dox according to an embodiment of the present invention was administered with FHSirp alpha oligonucleotide and doxorubicin. Cellular immune responses to CL25 cancer cells were investigated. That is, the level of interferon-gamma (INF-γ) of T cells specific to β-galactosidase, an antigen of CT26.CL25, was quantified by ELISA (R & D Systems, Inc, USA) analysis. 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 . The cells in the tubes were washed with PBS and suspended in RPMI 1640 medium to isolate tumor-drained lymph node (TDLN) cells and splenocytes. Separated TDLN cells and spleen cells were inoculated into 24-well plates at 1 10 ⁶ cells / 500 μl and 1 ㅧ 10 ⁷ cells / ml, respectively, and β-gal peptide (TPHPARIGL, 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 ) And P1A peptide (negative control) were treated at a concentration of 5 μg / ml for 24 hours to promote the activation of IFN-γ-secreting CD8 ⁺ T cells. The supernatant was then separated and quantified by ELISA (R & D Systems, Inc, USA) analysis of interferon-gamma (INF-γ).

As a result, as shown in FIGS. 4A and 4B, when the β-gal peptide and the AH1 peptide were treated with tumor-bearing lymph node cells and spleen immunocytes isolated from an animal to which FHSirpα-dox was administered according to an embodiment of the present invention , INF-y expression was significantly increased. In particular, the FHSirpα 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. Other drugs, such as the combination of other doxorubicin and recombinant Sirpα, showed little or no effect. That is, in the case of FHSirpα-dox, β-galactosidase protein is efficiently transferred to macrophages and dendritic cells to activate antigen-presenting cell function, and ultimately to effectively stimulate cytotoxic T cells, resulting in an immune response specific to CT26.CL26 cancer cells As a result,

1-4: Immunohistochemical analysis

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.

Specifically, the tumor tissues obtained from the FHSirpα-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 μm 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. After incubation with anti-CD8 antibody (BD Bioscience, USA) After washing, triamine signal amplification (TSA) was performed using a tyramine-conjugated flourophore (NEL794001KT, PerkinElmer, USA) and then washed three times to obtain a non-specific triamine- Eliminating Coupled Fluorescence Bonding After boiling in an aqueous solution of citric acid, DAPI was stained and photographed using a PerkinElmer Vectra platform.

As a result, as shown in Fig. 5, 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.

1-5: Antitumor effect in tumor implant

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.

Specifically, 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. On the 7th day after the injection of cancer cells, each test substance (buffer, wtFH-dox and FHSirpα -dox) was intraperitoneally administered once. At this time, the dose of each substance corresponds to wt FH-dox 15 mg / kg (corresponding to 1 mg / kg for doxorubicin content) and FHSirpα-dox 28 mg / kg (for doxorubicin 1 mg / kg ). Then, 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.

As a result, as shown in Fig. 6A, when doxorubicin was loaded on wild-type ferritin, the tumor growth rate was lower than that of the control group, but the tumor tissue still grew. On the other hand, in the case of FHSirp alpha-dox combined with Sirp alpha oligosaccharide and doxorubicin according to one embodiment of the present invention, cancer cells hardly grow even after 25 days. In addition, as shown in FIG. 6B, the weight of tumor tissue in the case of wtFH-dox was reduced to about 0.5 g, which was less than half of that of the control group exceeding 1.0 g, In the case of FHSirp? -Dox according to one embodiment, it could be said that there was almost no tumor. This demonstrates that the combination administration of Sirp alpha oligosaccharide and doxorubicin according to an embodiment of the present invention is more excellent in anti-cancer effect when administered in a tumor.

Experimental Example 2: Analysis of anticancer effect of FHSirp? -Dox

From the results of Experimental Examples 1-3, the inventors of the present invention will have a memory effect depending on the memory of immune cells since the action of the FHSirp? 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.

As a result, as shown in 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. In addition, as shown in FIG. 7C, in the FHSirpα-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 FHSirpα, survival rate after 80 days was 80%, which was better after FHSirpα-Dox, and survival rate after 80 days after doxorubicin alone or recombinant Sirpα alone was 50%.

The above results suggest that 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.

Experimental Example 3: Anticancer effect of FHSirp? -Dox on various tumors

3-1: Analysis of anticancer effect on CT26 cells

In order to confirm whether the combination administration of Sirp alpha oligonucleotide and doxorubicin according to an embodiment of the present invention shows an anticancer effect on cancer cells of different kinds, the present inventors used CT26 cells, which are general colon cancer cells not expressing? -Galatosidae, We analyzed the anticancer effect on the animal model animals.

Specifically, 8 weeks old 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, mSirpα + dox , FHSirpα-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). Then, the volume of cancer cells was measured at intervals of 3 days until the 25th day after the injection of the cancer cells by the method described in Experimental Example 1-1, and the tumor tissues of the experimental animals were extracted and weighed on the 25th day.

As a result, as shown in FIGS. 8A and 8B, inhibition of growth of cancer cells was insignificant in the case of dox, wtFH-dox and mSirpα + dox, whereas in the case of FHSirpα-dox according to one embodiment of the present invention, I was able to confirm that it was not nearly growing. In the case of dox and mSirpα + dox, the weight of tumor tissue was not significantly lower than that of the control group, and the weight of wtFH-dox was significantly (P <0.05) But the extent of it was weak. On the other hand, the FHSirp alpha-dox according to an embodiment of the present invention is almost free from tumor tissue.

3-2: Analysis of anticancer effect on B16F10-Ova cells

The present inventors analyzed the anticancer activity against B16F10-Ova cells, a kind of melanoma expressing ovalbumin.

Specifically, 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. On the 7th day after the injection of cancer cells, , wtFH-dox, mSirpα + dox, and FHSirpα-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). Then, the volume of cancer cells was measured at intervals of 3 days until the 25th day after the injection of the cancer cells by the method described in Experimental Example 1-1, and the tumor tissues of the experimental animals were extracted and weighed on the 25th day.

As a result, as shown in FIGS. 9A and 9B, inhibition of growth of cancer cells was insignificant in the case of dox, wtFH-dox and mSirpα + dox, whereas in the case of FHSirpα-dox according to one embodiment of the present invention, I was able to confirm that it was not nearly growing. In the case of dox, wtFH-dox and mSirpα + dox, tumor tissue weights were lower than those of the control group, but not significant. On the other hand, FHSirp? -Dox according to one embodiment of the present invention is almost free from the tumor tissue.

3-3: Analysis of cross-priming ability

The present inventors investigated whether the administration of Sirpa multimer and doxorubicin combination according to an embodiment of the present invention has a cross-priming ability of dendritic cells. 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.

Specifically, 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, FHSirpα-dox 28 mg / kg (1 mg / kg as doxorubicin) was injected intravenously three times in total every 3 days. 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 INF-γ was measured by ELISA analysis.

As a result, as shown in FIG. 10, INF-γ was secreted by 100 pg / ml or more in the FHSirpα-dox-treated group according to one embodiment of the present invention, whereas INF-γ expression was extremely small in the control group. This is because 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 .

 Then, the present inventors carried out further analysis as follows to confirm that the above phenomenon is a true cross-priming ability.

Specifically, 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, FHSirpα and FHSirpα-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 FHSirpα, and 28 mg / kg (1 mg / kg for dox) of FHSirpPα-dox. Finally, 10 days after intravenous administration, 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.

As a result, compared to the buffer, dox, and FHSirpa groups, as shown in FIGS. 11A and 11B, compared with the FHSirp? -Dox according to one embodiment of the present invention, injected CFSE-stained OT-1 T cells more effectively proliferated Trends. Specifically, in the case of FHSirpα-dox, the proportion of proliferated 7th generation OT-1 T cells was 15%, which was significantly increased compared with the other groups.

Experimental Example 4: Antitumor effect of FcSirp alpha + Mtx

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.

4-1: Analysis of phagocytosis by FcSirpα

To investigate the effect of FcSirpα on the phagocytic action of macrophages on cancer cells, the present inventors analyzed phagocytosis on cancer cells using bone marrow-derived macrophages (BMDM). For this purpose, BMDM was stained with 1 μM of CellTracker Green CMFDA (Thermo Fisher Scientific, USA). The stained macrophages of 20x10 ⁴ cells were inoculated into 35 mm confocal dishes with 2 ml of RPMI medium. 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 FcSirpα 5 &lt; / RTI &gt; or 400 nM of FHSirp alpha prepared in Example 1. At this time, FcSirpα 5 μM and FHSirpα 400 nM have the same amount of Sirpα. After 2 hours of co-cultivation, the degree of cancer cell phagocytosis was analyzed using a fluorescence microscope (Fig. 12A, red: cancer cell proliferated, green: bone marrow-derived macrophage) and quantified (Fig. As a result, as shown in FIGS. 12A and 12B, FcSirpα and FHSirpα significantly increased cancer cell phagocytic ability of macrophages, and FcSirpα showed the best phagocytic capacity.

The present inventors then analyzed the phagocytosis of cancer cells using a flow cytometer with different concentrations of FcSirpα in order to determine the concentration of FcSirpα phagocytic activity. For this purpose, BMDM was stained with 1 μM 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 HT29 cancer cell line stained with RPMI medium prepared with bone marrow-derived macrophages and 1 μM CellTracker Deep redd (Thermo Fisher Scientific, USA) was incubated for 2 hours at about 37 ° C. in the control or FcSirpα prepared in Example 1 1 &lt; / RTI &gt; At this time, FcSirpα 5 μM and FHSirpα 400 nM have the same amount of Sirpα. After 2 hours of co-culturing, the degree of cancer cells was analyzed using a flow cytometer. As a result, as shown in FIG. 12C, FcSirpα 50 nM, FcSirpα 500 nM, FcSirpα 5 μM and FHSirpα 400 nM, The cancer cell phagocytic ability was significantly increased, and especially FcSirpα showed an increase in the phagocytic ability from the concentration of 50 nM.

4-2: FcSirp &lt; + &gt; in vivo  Anticancer effect analysis

Based on the above experimental results, the present inventors investigated the synergistic effect of FcSirpα and mitoxantrone, which is an immunogenic cell death inducer, in combination.

Specifically, 5X10 ⁵ cells of B16F10 cancer cells were subcutaneously injected into C57BL / 6 wild-type mice by subcutaneous injection on the left side, and mitoxantrone (MTX) was administered 10 days after the injection of cancer cells on the 6th day ml or FcSirpa at a dose of 400 μg. In the case of FcSirpα + MTX group, mitoxantrone was administered by intra-tumor injection at a dose of 400 μg FcSirpα 4 h after 10 μg tumor injection. After the administration of the drug, 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). Experimental Results As shown in Figs. 13a to 13c, the MTX + FcSirpα group showed the best survival rate and anticancer effect.

4-3: Evaluation of CD8 T cell infiltration ability of FcSirpα + MTX

From the results of Experimental Example 4-2, the present inventors evaluated whether or not the adaptive immune response was also enhanced by the combination of FcSirpα + MTX by measuring the degree of infiltration of CD8 ⁺ T cells into the tumor microenvironment. Specifically, 5X10 ⁵ cells of B16F10 cancer cells were subcutaneously injected into C57BL / 6 wild type mice to induce cancer. On the 6th day after the injection of cancer cells (with the day of cancer cell injection on the 0th day), 10 μg of Mitoxantrone or FcSirpα 400 lt; RTI ID = 0.0 &gt; mg / ml. &lt; / RTI &gt; In the case of the FcSirpα group, mitoxantrone was administered by intratumoral injection at a dose of 400 μg FcSirpa 4 h after 10 μg tumor injection. On day 21 after the injection of cancer cells, the mice were sacrificed and the cancer tissues were extracted and made into single cells. After anti-CD8 antibody staining, the degree of CD8 ⁺ T cell infiltration in cancer tissues was analyzed by flow cytometry. As a result, as shown in Fig. 14, significant CD8 ⁺ T cell increase was observed in the FcSirp alpha MTX group as compared with the other groups. This indicates that the multimeric Sirp alpha fusion protein according to an embodiment of the present invention exhibits remarkable anticancer activity by enhancing the acquired immune response as well as the innate immune response upon administration of the immunogenic proliferation inducer.

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. Thus, when a concomitant use of a multimerization domain to mass-form a Sirp protein and combine with a detersive compound that induces immunogenic cell death such as doxorubicin is developed, it is possible to efficiently and effectively treat the intractable cancer It is expected that the development of therapeutic agents will be possible.

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), and 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), and 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.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, and that various modifications and equivalent embodiments may be made by those skilled in the art. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (38)

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 immunogenic apoptosis-inducing agent. The method according to claim 1, Wherein the signal regulatory protein is Sirp &lt; alpha &gt;, Sirp [gamma], or a high affinity variant thereof. The method according to claim 1, Wherein the fusion protein comprises the signal regulatory protein and the functional peptide. The method of claim 3, 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 ID = 0.0 &gt; and / or &lt; / RTI &gt; Wherein the signal regulatory protein is comprised of an amino acid sequence selected from the group consisting of SEQ ID NOS: 5, 7, 9, 11, 13, and 15 to 65. 5. The method of claim 4, Wherein the multimerization domain is a dimerization domain, a trimerization domain, a tetramerization domain, a hexamerization domain, a 12merization domain, or a 24merization domain. The method according to claim 6, The dimerization domain comprises an Fc fragment comprising the hinge region of the heavy chain of the antibody and the CH ₂ and CH ₃ moieties, the cytoplasmic domain of the receptor tyrosine kinase (RTK), the dimerization domain of kinesin, the dimerization domain of fibronectin, the Tol- (TLR) dimerization domain, or a dimerization domain of tubulin. The method according to claim 6, Wherein the trimerization domain is a trimerization domain of collagen, a trimerization domain of TRAIL, a trimerization domain of an Eml4 protein, or a trimerization domain of Clathrin. The method according to claim 6, Wherein the tetramerization domain is a tetramerization domain of p53, a tetramerization domain of DsRed, or a tetramerization domain of acetylcholinesterase (AChE). The method according to claim 6, Wherein said hexamerization domain is a hexamerization domain of HSP100 protein. The method according to claim 6, Wherein the 12-methylation domain is SPD (surfactant protein D, WO2013115608A1) or M. tuberoculosis Hsp16.3. The method according to claim 6, Wherein the 24- methylation domain is a ferritin heavy chain protein, a ferritin light chain protein, M. jannanschii Hsp16.5, or a yeast Hsp26. 3. The method of claim 2, Wherein the high-affinity mutant is a cancer treatment wherein at least one amino acid selected from the group consisting of the 6th, 27th, 41st, 47th, 53rd, 54th, 56th, 66th, A pharmaceutical composition. 5. The method of claim 4, The protein specifically binding to the cancer cell-specific receptor or ligand may include an antibody specifically binding to the cancer cell-specific receptor or ligand, a functional fragment thereof or an antibody analogue, and the cancer cell-specific receptor or ligand A pharmaceutical composition for treating cancer, which is a protein having a RGD domain specifically binding to a specifically binding protein, for example, an integrin specifically expressed in a cancer cell. 5. The method of claim 4, TIM1, TIM2, TIM3, TIGIT, CD226, CD160, CD20, CD20, CD30, CD30, CD27, SLAM, 2B4, NKG2D) LAG3, B7-1, B7-H1, GITR, HVEM, or OX40L, or a co-stimulatory domain thereof. The pharmaceutical composition according to claim 4, wherein the chemokine receptor is CCR or CXCR. 17. The method of claim 16, Wherein said CCR is CCR1, CCR2, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10. 17. The method of claim 16, Wherein the CXCR is CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CXCR6, or CXCR7. The pharmaceutical composition according to claim 4, wherein said apoptosis inducing ligand is TRAIL or FasL. The method according to claim 1, Wherein the signal transduction protein comprises a mutation of any one or more of the following: Sirp ?: V6I, V27I, A27I, I31F, E47V, K53R, E54Q, H56P, L66T, S66T, and V92I; And Sirp ?: M6I, V27I, L31F, E47V, L53R, E54Q, H56P, L66T, and Y92I. The method according to claim 1, Wherein the fusion protein further comprises a linker peptide between the signal regulatory protein and the functional peptide. 22. The method of claim 21, The linker peptides include (G ₄ S) n, (GSSGGS) n, KESGSVSSEQLAQFRSLD (SEQ ID NO: 5), EGKSSGSGSESKST (SEQ ID NO: 66), GSAGSAAGSGEF (SEQ ID NO: 67), (EAAAK) n, CRRRRRREAEAC (EAAAK) ₄ ALEA (EAAAK) ₄ A (SEQ ID NO: 69), GGGGGGGG (SEQ ID NO: 70), GGGGGG (SEQ ID NO: 71), GGGGS (SEQ ID NO: 72), AEAAAKEAAAAKA (Ala-Pro) n, VSQTSKLTRAETVFPDV (SEQ ID NO: 75), PLGLWA (SEQ ID NO: 76), TRHRQPRGWE (SEQ ID NO: 77), AGNRVRRSVG (SEQ ID NO: 78), RRRRRRRR (SEQ ID NO: 79), GFLG Or GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 81). The method according to claim 1, Wherein said fusion protein further comprises a tag peptide for purification at the N-terminus or C-terminus. 24. The method of claim 23,  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 method according to claim 1, The immunogenic cell death inducer may be selected from the group consisting of an anthracycline based chemotherapeutic agent, a taxane based chemotherapeutic agent, an anti-EGFR antibody, a BK channel agonist, bortezomib, cardiac glycoside, a cyclophosphamide anticancer agent, a GADD34 / PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone or oxaliplatin. 26. The method of claim 25, The anthracycline anticancer agent may be at least one selected from the group consisting of daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or valrubicin ). &Lt; / RTI &gt; 26. The method of claim 25, Wherein said anti-EGFR antibody is cetuximab. 26. The method of claim 25, Wherein said anhydrous glycosides are used in combination with non-immunogenic apoptosis inducing agents. 26. The method of claim 25, Wherein said GADD34 / PP1 inhibitor is used in combination with mitomycin. 26. The method of claim 25, Wherein said taxane-based anticancer agent is paclitaxel or docetaxel. The method according to claim 1, A pharmaceutical composition for the treatment of cancer, further comprising an immune checkpoint inhibitor. 32. The method of claim 31, Wherein said immune checkpoint inhibitor is a PD-1 / PD-L1 interaction inhibitor or a CTLA-4 / B7-1 / B7-2 interaction inhibitor. 32. The method of claim 31, Wherein said PD-1 / PD-L1 interaction inhibitor is an antibody targeting PD-1 or PDL1 or a functional fragment or single chain-based antibody analogue of said antibody. 34. The method of claim 33, Wherein the antibody targeting PD-1 or PDL1 is Pembrolizumab, Nivolumab, Atezolizumab or Avelumab. 33. The method of claim 32, The CTLA-4 / B7-1 / B7-2 interaction inhibitor may be an antibody targeting CTLA-4, B7-1 or B7-2 or a functional fragment of the antibody or a single chain- A pharmaceutical composition. 36. The method of claim 35, Wherein the CTLA-4 / B7-1 / B7-2 interaction inhibitor is Ipilimumab. 34. The method according to claim 33 or 35, The single-stranded-based antibody analog may be a single-chain-based antibody analog such as scFv, sdAb, diabody, monobody, variable lymphocyte receptor (VLR), nanobody, Lt; _{RTI ID = 0.0} &gt; (VH _{&lt; /} RTI _&gt; H). Use of a signal-regulatory protein or a fusion protein comprising the signal-regulating protein and an immunological cell death inducer in the preparation of a pharmaceutical composition for the treatment of cancer.

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