KR102579687B1 - A novel pharmaceutical composition for treating cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment of cancer. The present invention relates to a signal-regulatory protein or a fusion protein containing the signal-regulatory protein, and a multimer and immunogenicity of the fusion protein containing a multimerization domain. Provided is a pharmaceutical composition for treating cancer containing an apoptosis-inducing agent as an active ingredient.

Description

A novel pharmaceutical composition for treating cancer}

The present invention relates to a novel composition for treating cancer.

Methods for treating cancer include surgery, radiation therapy, and anticancer drug administration, but these treatment methods involve side effects or are limited in application depending on the progress of the cancer. In particular, as a result of repeated research, the types of anticancer drugs have increased in quantitative terms, but there has been no significant change in qualitative terms. This is because most anticancer drugs work as a mechanism to stop the cell cycle of actively dividing cells and cause them to die. As a result, they attack normally dividing cells in addition to cancer cells, causing hair loss, loss of appetite, and reduction of white blood cells, which are typical side effects of anticancer drugs. A decrease in immunity occurs. Doxorubicin, a representative anticancer drug, is an anticancer drug belonging to the anthracycline class of antitumor drugs. Anthracycline class anticancer drugs are anticancer drugs that act cell cycle selectively, inhibiting cell division, malignant lymphoma (lymphosarcoma, Hodgkin's disease and non-Hodgkin's disease), It is used to treat various cancers, including digestive cancer (stomach cancer, liver cancer, rectal cancer, gallbladder and bile duct cancer, colon cancer, and pancreatic cancer), acute myeloid leukemia, soft tissue osteosarcoma, breast cancer, ovarian cancer, lung cancer, bronchial cancer, bladder cancer, and Wilm's tumor. According to recent research results, anthracycline anticancer drugs have been reported to induce immunogenic apoptosis of cancer cells by inducing preapoptotic translocation of caleticulin to the cell membrane (Obeid et al . , Nat. Med. , 13(1): 54-61, 2007). Meanwhile, the above-mentioned cancer treatment strategy through strengthening immune function has been attracting attention recently. Research on the correlation between immune cells and cancer began worldwide in the 1970s, and has been studied to identify immune cells, which are biological weapons that can fight cancer. As functions have become very important, research reports have increased exponentially since 2000, and anticancer immunotherapy, such as Keytruda (Pembrolizumab, developed by Merck), an anticancer immunotherapy antibody, received accelerated approval through the US FDA in September 2014. The importance of research is emerging. In particular, since immune cells patrol and search for cancer cells, the effect is not limited to the local area, but not only monitors and suppresses the development of cancer throughout the body, but can also be applied to a variety of cancer cells. Therefore, there is a need to move away from existing approaches that focus on cancer cell necrosis and propose a new paradigm for cancer prevention and treatment strategies that overcome the cancer microenvironment by controlling the dynamic networking of immune cells in the human body.

The present invention is intended to solve various problems including the above problems, and provides an immunotherapeutic agent and a use thereof that can maximize the efficiency of cancer immunotherapy through inducing immunogenic cell death and controlling immune cell networking. The purpose. However, these tasks are illustrative and do not limit the scope of the present invention.

According to one aspect of the present invention, Sirp high affinity variants and antibodies containing one or more substitution mutations selected from the group consisting of V6I, V27I, A27I, I31F, E47V, K53R, E54Q, H56P, L66T, S66Y and V92I. Provided is a pharmaceutical composition for cancer treatment comprising a fusion protein containing an Fc fragment containing a heavy chain hinge region and CH2 and CH3 regions and an anthracycline-based anticancer agent as active ingredients.

According to one aspect of the present invention, Sirp high affinity variants and antibodies containing one or more substitution mutations selected from the group consisting of V6I, V27I, A27I, I31F, E47V, K53R, E54Q, H56P, L66T, S66Y and V92I. A cancer treatment method comprising administering to an individual in need of treatment a fusion protein containing an Fc fragment including the heavy chain hinge region and CH2 and CH3 regions and an anthracycline-based anticancer agent. This is provided.

According to one embodiment of the present invention as described above, the present invention can maximize the cancer treatment effect through the synergistic effect of combined administration of a signal regulatory protein and an immunogenic apoptosis inducing agent.

Figure 1a is a schematic diagram showing the schematic appearance of a doxorubicin-loaded Sirp protein multimer loaded with doxorubicin inside a nanocage created by self-assembly of a fusion protein composed of ferritin heavy chain protein and Sirpα according to an embodiment of the present invention. 1B is a graph showing the results of fluorescence FPLC analysis of a FHSirpα multimer (FHSirpα-dox) loaded with doxorubicin according to an actually manufactured embodiment of the present invention, and Figure 1C is an actually manufactured example of the present invention. It is a histogram showing the results of analyzing the particle size of the doxorubicin-loaded FHSirpα multimer (FHSirpα-dox) through dynamic light scattering (DLS) analysis, and Figure 1d shows the doxorubicin-loaded FHSirpα multimer (FHSirpα-dox). This photo was taken with a transmission electron microscope.
Figure 2a is a schematic diagram showing the experimental schedule of the animal test for analyzing the in vivo anticancer activity of Experimental Example 1-1, and Figure 2b is a graph showing the results of the in vivo anticancer effect analysis performed according to the schedule in Figure 2a ( ^* : P < 0.05, ^*** : P < 0.001), Figure 2c is a photograph of the tumor area of each experimental group on day 25 of cancer cell inoculation (left and right sides represent independent experimental animals from the same experimental group), and Figure 2d is a photograph of the tumor area of each experimental group on day 25 of cancer cell inoculation. This is a graph showing the results of measuring the weight of tumor tissue extracted from each experimental group on day 1 ( ^*** : P < 0.001).
Figure 3a is an anti-F4/80 antibody to confirm the presence and ratio of macrophages and dendritic cells in tumor tissues extracted from cancer model animals administered FHSirpα-dox and a buffer solution as a control group according to an embodiment of the present invention, respectively. and a histogram showing the results of FACS analysis using an anti-CD11c antibody, and Figure 3b shows the ratio of F4/80-positive cells (left) and CD11c-positive cells (right) compared to the total number of cells isolated from tumor tissue. This is a graph showing ( ^* : P < 0.05, ^*** : P < 0.001).
Figure 4a shows various effects on isolated tumor drained lymph node cells after inoculation of CT26.CL25 cancer cells in experimental animals and administration of buffer or various test substances (dox, wrFH-dox, mSirpα + dox, and FHSirpα-dox). It is a graph showing the results of measuring the expression level of INF-γ upon treatment with peptides (β-galactosidase, gp70-derived AH1 peptide and PIA peptide) ( ^* : P < 0.05, ^** : P < 0.01), and Figure 4b is the above. Figure 4a is a graph measuring the expression level of INF-γ when spleen cells isolated from the experimental animals were treated with the functional peptide ( ^* : P < 0.05, ^** : P < 0.01).
Figure 5 is an immune tissue showing the results of comparing the degree of accumulation of CD8 ⁺ T cells at the tumor site after tumor transplantation in experimental animals and administration of buffer or doxorubicin combination drug (FHSirpα-dox) according to an embodiment of the present invention. As a result of chemical analysis, the top is a fluorescent staining photo of a tumor tissue section after administration of a buffer solution, the bottom is a fluorescent staining photo of a tumor tissue section after administration of FHSirpα-dox according to an embodiment of the present invention, and the leftmost column is DAPI. shows the results of staining the nucleus using, the second column from the left shows the results of staining using an anti-CD8 antibody, and the third column from the left is a merged image of the DAPI staining and the anti-CD8 antibody staining results. , and the rightmost column shows the results of cell segmentation analysis.
Figure 6a shows the tumor size over time after intratumoral administration of buffer, wtFH-dox, and FHSirpα-dox according to an embodiment of the present invention to a cancer model animal inoculated with CT26.CL25 cancer cells. It is a graph showing the results ( ^** : P < 0.01, ^*** : P < 0.001), Figure 6b is a photograph of the tumor inoculation site of the cancer model animal 25 days after cancer cell inoculation, and Figure 6c is the upper figure. This is a graph showing the results of measuring the weight of tumor tissue extracted from the experimental animal in 6a on the 25th day of cancer cell inoculation ( ^* : P < 0.05, ^** : P < 0.01).
Figure 7a shows that after administration of buffer or various experimental substances (dox, wtFH-dox, mSirpα + dox, and FHSirpα-dox) to cancer model animals by CT26.CL25 cancer cell inoculation, tumor tissue was extracted on the 25th day of cancer cell inoculation and the corresponding area was removed. After suturing, the opposite side was rechallengeed by inoculating 1x10 ⁶ cells, the same amount as the CT26.CL25 cancer cells initially inoculated, and then secondary cancer cell inoculation sites were examined over time for the control group and the FHSirpα-dox treatment group. It is a series of photos taken (the top and bottom are photos of the same animal taken at different shooting angles), and Figure 7b is a graph measuring the ratio of tumor-free animals over time in the cancer model animals, Figure 7c is a graph measuring the survival rate of the cancer model animals over time.
Figure 8a shows the tumor tissue after intravenous administration of buffer or various test substances (dox, wtFH-dox, mSirpα + dox, and FHSirpα-dox) to cancer model animals by CT26 cancer cell inoculation, at 3-day intervals until the 25th day of cancer cell inoculation. This is a graph showing the results of measuring the size ( ^*** : P < 0.001), and Figure 8b is a graph showing the results of measuring the weight of the tumor tissue extracted on the 25th day of cancer cell inoculation in the cancer model animal ( ^* : P < 0.05, ^*** : P < 0.001).
Figure 9a shows tumor growth at 3-day intervals until the 25th day of cancer cell inoculation after intravenous administration of buffer or various test substances (dox, wtFH-dox, mSirpα + dox, and FHSirpirpα-dox) to cancer model animals by B16F10-Ova cancer cell inoculation. This is a graph showing the results of measuring the size of the tissue ( ^** : P < 0.01), and Figure 9b is a graph showing the results of measuring the weight of the tumor tissue extracted on the 25th day of cancer cell inoculation in the cancer model animal ( ^** : P < 0.01).
Figure 10 shows that after administering buffer or FHSirpα-dox to a cancer model animal by inoculating B16F10-Ova cancer cells, the tumor tissue was extracted and converted into single cells, and the isolated dendritic cells were co-cultured with OT-1 T cells and then added to the culture supernatant. This graph confirms the cross-priming ability by quantifying the amount of INF-γ present ( ^* : P < 0.05).
Figure 11a shows that after administration of buffer or various test substances (dox, FHSirpα and FHSirpα-dox) to a cancer model animal inoculated with the B16F10.Ova cancer cells, OT-1 T-cells stained with CFSE were injected into the cancer model animal. It is a histogram showing the results of FACS analysis after single-celling the extracted tumor-draining lymph node cells, and Figure 11b shows the ratio (%) by generation of OT-1 T-cells stained with CFSE for the results of Figure 11a. This graph represents ( ^** : P < 0.01).
FIG. 12A is a series of fluorescence microscopy images analyzing the effect of FHSirpα and FcSirpα on the phagocytosis of BMDM against cancer cells (HT29) according to an embodiment of the present invention, and FIG. 12B is the cancer cell phagocytosis rate of BMDM of FIG. 12A. It is a graph showing the results of quantifying ( ^* : P <0.05; ^*** : P < 0.001), and Figure 12c shows various concentrations (50 nM, 500 nM, and 5 μM) of FcSirpα and 400 nm of FHSirpα in BMDM. This is a graph showing the degree of phagocytosis on HT29 cancer cells during treatment ( ^*** : P < 0.001).
Figure 13a shows tumor tissue over time after intratumoral injection of FcSirpα and/or mitoxantrone according to an embodiment of the present invention to C57BL/6 wild-type mice in which cancer was induced by subcutaneous injection of B16F10 cancer cells. It is a graph showing the results of measuring the volume ( ^*** : P < 0.001), and Figure 13b is a graph recording the survival rate of the experimental animals used in the experiment of Figure 13a up to 21 days after injection of cancer cells ( ^{* **} : P < 0.001), Figure 13c is a graph showing the results of measuring the average weight of the cancer tissue extracted after the experimental animal was sacrificed on the 21st day after cancer cell injection, and Figure 13d is the change in body weight of the experimental animal. This is a graph showing the measurement results.
Figure 14 shows the experimental animals on the 21st day after injection of the cancer cells after intratumoral injection of FcSirpα and/or mitoxantrone according to an embodiment of the present invention to C57BL/6 wild-type mice in which cancer was induced by subcutaneous injection of B16F10 cancer cells. This is a graph showing the results of measuring the degree of CD8 ⁺ T cell infiltration by single-celling the cancer tissue extracted after sacrifice and performing flow cytometry using an anti-CD8 antibody ( ^* : P < 0.05).

Definition of Terms

As used in this document, “immunogenic cell death” refers to a type of cell death caused by cytostatics such as anthracyclines, oxaliplatin, and bortezomib, or by radiotherapy and photodynamic therapy. It means cell death. Unlike general cell death, the immunogenic cell death of cancer cells can induce an effective anti-cancer immune response through activation of dendritic cells and subsequent activation of specific T cell responses. Substances that cause immunogenic cell death are called “immunogenic cell death inducers.” The immunogenic cell death and immunogenic cell death-inducing agents are well summarized in Kroemer et al. ( Annu. Rev. Immunol ., 31: 51-72, 2013). The above document is incorporated herein by reference in its entirety.

The term “multimerization domain” used in this document refers to a protein domain containing a minimum region that plays an important role in multimerizing the same type of protein through self-assembly. The multimerization domain includes domains involved in self-assembly of various self-assembly proteins, such as a dimerization domain, trimerization domain, tetramerization domain, hexamerization domain, 12merization domain, and 24merization domain. The dimerization domain specifically includes an Fc fragment containing the hinge region and CH2 and CH3 portions of the heavy chain of the antibody, the cytoplasmic domain of receptor tyrosine kinase (RTK), the dimerization domain of kinesin, the dimerization domain of fibronectin, and the Tol-like receptor. (TLR) dimerization domain, tubulin dimerization domain, etc. The trimerization domain includes the trimerization domain of collagen, the trimerization domain of TRAIL, the trimerization domain of Eml4 protein, and the trimerization domain of Clathrin. The tetramerization domain includes the tetramerization domain of p53, the tetramerization domain of DsRed, and the tetramerization domain of acetylcholine esterase (AChE). The hexamerization domain includes the hexamerization domain of the HSP100 protein, and the 12merization domain includes SPD (surfactant protein D, WO2013115608A1) and M. tuberoculosis Hsp16.3. The 24-merization domains include ferritin heavy chain protein, ferritin light chain protein, M. jannanschii Hsp16.5, and yeast Hsp26. Proteins that multimerize through self-assembly as described above are called “self-assembly proteins,” and the self-assembly proteins form multimers through regular arrangement upon expression without the help of special inducers, thereby forming nanoparticles. It refers to a protein that can be formed. Self-assembling proteins include small heat shock protein (sHsp), ferritin, vault, P6HRC1-SAPN, M2e-SAPN, MPER-SAPN, and various viral or bacteriophage capsid proteins. The self-assembling protein is well described in Hosseinkhani et al. ( Chem. Rev. , 113(7): 4837-4861, 2013). The above document is incorporated herein by reference in its entirety.

The term "Sirp (signal-regulated protein)" used in this document is a regulatory membrane glycoprotein among the Sirp family of proteins that is mainly expressed in bone marrow cells and in stem cells or nerve cells. Among the Sirps, Sirpα and Sirpγ act as inhibitory receptors and interact with CD47 protein, a widely expressed transmembrane protein, which is called the "don't eat me" signal. These interactions negatively regulate the effector functions of innate immune cells, such as host cell phagocytosis. This is similar to the self-signaling provided by MHC I family molecules via Ig-like or Ly49 receptors. Cancer cells overexpressing CD47 activate Sirpα or Sirpγ to inhibit macrophage-mediated destruction. According to a recent study, there is a report that a high-affinity variant of Sirpα increases phagocytosis of cancer cells by masking CD47 on cancer cells (Weiskopf et al ., Science 341(6141): 88-91, 2013).

The term "ferritin heavy chain protein (hereinafter abbreviated as 'FH')" used in this document refers to a protein that constitutes the heavy chain subunit of ferritin, a major intracellular iron storage protein in prokaryotes and eukaryotes. , the ferritin protein consists of 24 subunits each of the ferritin heavy and light chains. The main function of the ferritin protein is to store iron in a soluble, non-toxic state. It is known that 24 subunits of ferritin heavy chain protein self-assemble to form hollow nanoparticles even without light chain protein (Cho et al ., Biochem. Biophys. Res. Commun . 327(2): 604-608, 2005) . Ferritin heavy chain protein can play the role of a nanocage by loading other drugs into the empty internal space inside self-assembled nanoparticles, and due to this characteristic, it is being studied for purposes such as drug delivery vehicles.

The term "anthracyclin-type anticancer agent" used in this document refers to Streptomyces peucetius var. caesius refers to a class of cell cycle non-specific anticancer drugs used for cancer chemotherapy. Anthracycline-based anticancer drugs are used to treat various cancers, including leukemia, lymphoma, breast cancer, stomach cancer, uterine cancer, ovarian cancer, bladder cancer, and lung cancer, and are one of the most effective anticancer drugs among chemotherapy drugs that have been developed conventionally. The first anthracycline anticancer drug discovered was daunorubicin, followed by doxorubicin, followed by epirubicin, idarubicin, and pixantrone. , sabarubicin, valrubicin, etc. exist. The mechanism of action of anthracycline anticancer drugs is to inhibit DNA and RNA synthesis by inserting between base pairs of DNA/RNA strands, thus interfering with replication of rapidly growing cancer cells, and to inhibit topoisomerase II enzyme activity, resulting in supercoiling. Interfering with transcription and replication by inhibiting DNA relaxation, inducing damage to DNA, proteins and cell membranes through the formation of iron-mediated free oxygen radicals and DNA damage response, chromatin deregulating the epigenome and transcriptome. Induction of histone eviction has been discussed. Recent studies have reported that doxorubicin increases Th1 immune responses by activating CD4 ⁺ cells (Park et al ., Int. Immunopharmacol . 9(13-14): 1530-1539, 2009) and dendritic cells (dendritic cells). It has been reported that the combined administration of doxorubicin and doxorubicin exhibits anticancer activity by inducing immunogenic cell death of osteosarcoma (Kawano et al ., Oncol. Lett . 11:2169-2175, 2016).

The term "taxanoid anticancer agent or taxne anticancer drug" used in this document refers to diterpenoid taxane derivatives extracted from the plant Taxus sp., which refers to the structure of intracellular microtubules. It is a mitotic inhibitor with a mechanism that promotes assembly and inhibits disassembly. Currently commercialized drugs include paclitaxel and docetaxel, of which paclitaxel is a taxane-based anticancer drug extracted from the pericarp of the yew tree ( Taxus brevifolia ) and was approved by the U.S. FDA in 1992 as a treatment for incurable ovarian cancer. Docetaxel is a taxane-based anticancer drug derived from Taxus bacaata that has similar efficacy to paclitaxel. It is used to treat breast cancer, non-cell lung cancer, lymphoma, and bladder cancer, and has higher hydrophilic properties than paclitaxel. Recently, it has been revealed that taxane-based anticancer drugs also have a mechanism to promote immunogenic cell death of cancer cells by sensitizing them to cytotoxic T lymphocytes.

As used in this document, the term “immune checkpoint inhibitor” refers to a type of drug that blocks certain proteins produced by certain types of immune system cells, such as T lymphocytes, and some cancer cells, which stimulate the immune response. Inhibits and prevents T lymphocytes from killing cancer cells. Therefore, when these proteins are blocked, the “brakes” on the immune system are released and T lymphocytes are better able to kill cancer cells. The “immune checkpoints” that are well known to date include PD-1/PD-L1 and CTLA-4/B7-1/B7-2. PD-1 inhibitors include Pembrolizumab (brand name: Keytruda) and Nivolumab (brand name: Opdivo), and PD-L1 inhibitors, which are PD-1 ligands, include Atezolizumab (brand name: Tecentriq) and Avelumab (brand name: Bavencio). etc. exist. Meanwhile, CTLA-4 inhibitors that inhibit the interaction of CTLA-4/B7-1/B7-2, such as Ipilimumab (brand name: Yervoy), have been approved by the FDA. In recent years, it has achieved impressive success, particularly in patients with metastatic melanoma or Hodgkin lymphoma, and is showing great promise in clinical trials in patients with other types of cancer.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect of the present invention, Sirp high affinity variants and antibodies containing one or more substitution mutations selected from the group consisting of V6I, V27I, A27I, I31F, E47V, K53R, E54Q, H56P, L66T, S66Y and V92I. Provided is a pharmaceutical composition for cancer treatment comprising a fusion protein containing an Fc fragment containing a heavy chain hinge region and CH2 and CH3 regions and an anthracycline-based anticancer agent as active ingredients.

In the pharmaceutical composition for treating cancer, the Sirp high-affinity variant may include all of the substitution mutations of V6I, A27I or V27I, I31F, E47V, K53R, E54Q, H56P, L66T or S66T, and V92I.

In the pharmaceutical composition for treating cancer, the Sirp high-affinity variant may consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 15, 17, 18, and 23 to 33.

In the pharmaceutical composition for treating cancer, a linker peptide may be further included between the signal regulation protein and the Fc fragment.

As used herein, “antibody”, also called immunoglobulin, refers to a Y-shaped protein produced from plasma cells that is used by the immune system to identify or neutralize foreign substances such as bacteria or viruses. As used herein, the antibodies include various “functional fragments” derived from antibodies, such as Fab, F(ab')2, Fab', ScFv, and sdAb.

The term "functional fragment of an antibody" used in this document refers to a fragment derived from an antibody that has the ability to bind to an antigen and includes both fragments produced by cleaving an antibody with a protein cleavage enzyme as well as single-chain fragments produced by recombinant methods.

The term "Fab" used in this document is an antigen-binding antibody fragment, which is a fragment produced by chopping an antibody molecule with papain, a proteolytic enzyme, and is a dimer of two peptides, VH-CH1 and VL-CL. , other fragments produced by papain are referred to as Fc (fragment crystallizable).

The term "F(ab')2" used in this document is a fragment containing an antigen-binding site among fragments produced by cleaving an antibody with the proteolytic enzyme pepsin, and is in the form of a tetramer in which two Fabs are linked by a disulfide bond. indicates. Another fragment produced by pepsin is referred to as pFc'.

The term "Fab'" used in this document is a molecule with a similar structure to Fab, which is produced by isolating F(ab')2 under mild reducing conditions.

The term "ScFv" used in this document is an abbreviation for "single chain variable fragment" and is not an actual antibody fragment. The heavy chain variable region (VH) and light chain variable region (VL) of the antibody are divided into about 25 a.a. It is a type of fusion protein manufactured by linking with a large linker peptide and is known to have antigen-binding ability even though it is not an original antibody fragment (Glockshuber et al., Biochem. 29(6): 1362-1367, 1990).

The term “sdAb (single domain antibody)” used in this document refers to a nanobody, which is an antibody fragment composed of a single variable region fragment of an antibody. Although sdAbs derived from heavy chains are mainly used, single variable region fragments derived from light chains are also reported to bind specifically to antigens.

“Antibody mimetic” as used in this document is the minimum unit that maintains antigen-binding ability, unlike a typical full-length antibody in which two heavy chains and two light chains form a quaternary structure of a heterotetramer to exert its function. Fragments containing (e.g., Fab, F(ab')2, Fab' or single-chain variable fragment (scFv), which is an artificial fragment in which the variable regions of the heavy and light chains are connected by a linker, without the light chain A concept that includes antibody fragments (V _H H, V _NAR , etc.) derived from camelids or cartilaginous fish consisting of only heavy chains, or antibody-like proteins produced from protein scaffolds derived from non-antibodies such as nanobodies, monobodies, and variable lymphocyte receptors (VLR). am.

In the pharmaceutical composition for treating cancer, the linker peptide is (G ₄ S)n, (GSSGGS)n, KESGSVSSEQLAQFRSLD (SEQ ID NO: 5), EGKSSGSGSESKST (SEQ ID NO: 66), GSAGSAAGSGEF (SEQ ID NO: 67), (EAAAK) n, CRRRRRREAEAC (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 (SEQ ID NO: 72), AEAAAKEAAAAKA (SEQ ID NO: Number 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 (SEQ ID NO: 79), GFLG (SEQ ID NO: 80), or GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 81).

In addition, in the pharmaceutical composition, the fusion protein may additionally contain a tag peptide for purification at the N-terminus or C-terminus for efficient purification. The tag peptides include HisX6 peptide (SEQ ID NO: 82), GST peptide, FLAG peptide (DYKDDDK, SEQ ID NO: 83), streptavidin binding peptide, V5 epitope peptide (GKPIPNPLLGLDST, SEQ ID NO: 84), and Myc peptide (EQKLISEE, SEQ ID NO: 85). ), or it may be an HA peptide (YPYDVPDYA, SEQ ID NO: 86).

In the pharmaceutical composition for treating cancer, the anthracycline-based anticancer agents include daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, and savarubicin. It may be sabarubicin, mitoxantrone, or valrubicin.

The pharmaceutical composition for treating cancer may further include an immune checkpoint inhibitor, wherein the immune checkpoint may be PD-1, PD-L1, CTLA-4, B7-1, or B7-2, and the immune checkpoint inhibitor may be 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, a functional fragment of the antibody, or a single chain-based antibody analog, and the CTLA- The 4/B7-1/B7-2 interaction inhibitor may also be an antibody targeting CTLA-4, B7-1 or B7-2, a functional fragment of the antibody, or a single chain-based antibody analog, and the PD- 1 or the antibody targeting PDL1 may be Pembrolizumab, Nivolumab, Atezolizumab, or Avelumab, and the CTLA-4/B7-1/B7-2 The interaction inhibitor may be Ipilimumab.

In the pharmaceutical composition for treating cancer, the single chain-based antibody analog includes scFv, sdAb, diabody, monobody, variable lymphocyte receptor (VLR), and nanobody. or a camelid heavy chain fragment (V _H H).

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral formulations, but is preferably in a parenteral formulation. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. It is prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical composition is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories. It can have any one formulation.

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 administration, intraperitoneal administration, and transdermal absorption. .

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

As used in this document, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the individual, age, gender, drug activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concurrently used drugs, and other factors well known in the field of medicine. The pharmaceutical composition of the present invention can be administered at a dosage of 0.1 mg/kg to 1 g/kg, and more preferably at a dosage of 1 mg/kg to 500 mg/kg. Meanwhile, the dosage may be appropriately adjusted depending on the patient's age, gender, and condition.

The pharmaceutical composition of the present invention can be administered as an individual treatment or in combination with other anticancer agents, and can be administered sequentially or simultaneously with other conventional anticancer agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art.

According to another aspect of the present invention, the use of a signal-regulatory protein or a fusion protein containing the signal-regulatory protein and an immunogenic apoptosis inducing agent in the production of a pharmaceutical composition for treating cancer is provided. .

According to another aspect of the present invention, administering a multimer of a signal-regulatory protein or a fusion protein containing a multimerization domain and an immunogenic apoptosis inducer to an individual in need of treatment. A cancer treatment method comprising administering to the subject is provided.

The immunogenic apoptosis inducer can be loaded into the nanocage when the multimer of the fusion protein forms a nanocage. The loading of the immunogenic apoptosis inducer can be carried out using recombinant exosomes in a cell culture medium in which these drugs are dissolved. This can be achieved by cultivating cells that have been genetically engineered to produce, and the separated nanocage can be created by adding it to a solvent in which an anthracycline-based anticancer drug is dissolved and stirring it. Preferably, after forming a complex of a divalent metal ion (e.g., Cu ²⁺ , Fe ²⁺ , and Zn ²⁺ ) and an anthracycline-based anticancer agent in advance, the divalent metal is added to the internal pores of the prepared ferritin medium chain nanocage. It can be loaded by reacting in a buffer solution to allow the ion-anthracycline anticancer drug complex to permeate. In addition, anticancer drugs can be loaded into the internal pores through the disassembly-reassembly process of the ferritin medium chain nanocage due to pH differences, and anticancer drugs can be loaded into the protein nanocages through the pore opening phenomenon due to ion concentration differences. You can.

Recently, the identification of mutated proteins in tumors known as neoantigens, which are targets for cancer immunotherapy, has led to the development of treatments that increase anti-tumor T cell responses and, despite the highly mutagenic nature of cancer cells, Only 1% of expressed mutant proteins trigger an immune response in cancer patients. In order for a neoantigen to be immunogenic, it must be processed and presented by major histocompatibility complex (MHC) molecules on the cell surface, and efficient transfer of the immunogenic neoantigen from the tumor to the host's T cells is activated, which is activated by antigen-presenting cells. This is achieved only by neoantigenic peptides loaded with (APCs). We developed a novel strategy to overcome the activation-energy threshold of the immunosuppressive tumor microenvironment and mediate the delivery and presentation of tumor neoantigens by APCs to host T cells. The strategy is based on naturally derived ferritin-based nanocapsules containing drugs that induce immunogenic cancer cell death (ICD) as well as delivering ligands that enhance cancer cell phagocytosis by APC. Accordingly, the present inventors have developed an anticancer composite nanocage loaded with doxorubicin inside a nanocage created by self-assembly of a fusion protein composed of ferritin heavy chain protein and Sirpα, or a specific type of immune system cell such as T lymphocyte in the anticancer composite nanocage. and immune checkpoint inhibitors, which refers to a type of drug that blocks specific proteins produced by some cancer cells to induce immunogenic cell death against cancer cells, thus promoting tumor immunity specific to cancer antigens. developed a next-generation anticancer treatment that has no side effects caused by conventional anticancer drugs and has a sustainable anticancer effect by immune cells even after treatment.

Hereinafter, the present invention will be described in detail with reference to the attached drawings. For convenience of explanation, the sizes of components may be exaggerated or reduced in the drawings.

Figure 1a is a schematic diagram showing the schematic appearance of an anticancer composite nanocage loaded with doxorubicin inside the nanocage created by self-assembly of a fusion protein composed of ferritin heavy chain protein and Sirpα according to an embodiment of the present invention. As shown in Figure 1a, when a fusion protein linking the Sirpα or Sirpγ protein to the C-terminus of the ferritin heavy chain protein is expressed, an empty ferritin nanocage is formed through self-assembly of the ferritin heavy chain protein 24 subunits. When a complex of a drug and a metal particle such as copper (Cu) is processed in such a nanocage, the drug may be loaded into the nanocage. As for the FHSirpα nanocage loaded with doxorubicin produced in this way, it was confirmed that doxorubicin was properly loaded inside the nanocage as a result of fluorescence FPLC analysis (see Figure 1b), and dynamic light scattering analysis and electron microscopy showed that it was a spherical shape with a diameter of about 20 nm. It was confirmed that nanoparticles were formed (see Figures 1c and 1d)

As a result of in vivo anticancer activity analysis of doxorubicin-loaded multimeric FHSirpα prepared as described above in cancer model animals, it was confirmed that both intravenous and intratumoral administration significantly inhibited the growth of cancer cells (Figures 2a to 2a 2c, see Figures 6a to 6c).

The combined administration of multimeric Sirpα protein and doxorubicin according to an embodiment of the present invention as described above not only infiltrates macrophages, dendritic cells, and CD8 ⁺ cells, which are innate immune cells, into tumor tissue (FIGS. 3A, 3B, and FIG. 5), it was confirmed that cancer-specific immunity was acquired by activating immune cells present in the lymph nodes and spleen around the tumor tissue (Figure 4).

In addition, as a result of a rechallenge experiment in which tumor tissue was extracted from experimental animals administered the combination of multimeric Sirpα protein and doxorubicin of the present invention and then reinoculated with the same cancer cells, the recurrence of the same cancer was very efficiently suppressed without additional administration. It was found that (see FIGS. 7A to 7C).

In addition, the combined administration of multimeric Sirpα protein and doxorubicin according to an embodiment of the present invention has very effective anticancer activity not only in CT26.CL25 colon cancer cells, but also in CT26 colon cancer cells and B16F10-Ova melanoma cells. shown (Figures 8a to 9b).

In particular, in relation to the mechanism of anticancer activity against B16F10-Ova melanoma cells, it was confirmed that the combined administration of multimeric Sirpα protein and doxorubicin of the present invention has cross-priming ability, and as a result, it was found to have a cross-priming ability and was found to be naive with MHC type 1 molecules. It was confirmed that presenting antigen to CD8 ⁺ T cells promoted stimulation and differentiation of naive CD8 ⁺ T cells (FIGS. 10 to 11B).

The above results show that the combined administration of a multimeric Sirp protein and an immunogenic apoptosis inducing agent according to an embodiment of the present invention results in an anti-cancer based immune response that is very weak when the Sirp protein is administered alone and the immunogenic apoptosis inducing agent is administered alone. This suggests that it can have a very surprising synergistic effect on activity.

The combination drug of the present invention delivers drugs that induce immunogenic cancer cell death (ICD) as well as ligands that enhance cancer cell phagocytosis by antigen presenting cells (APCs). The combined administration of the present invention induces the secretion of danger signals and neoantigens from dying cancer cells, enhances tumor cell phagocytosis, and induces tumor-specific T cells by dendritic cells loaded with neoantigen peptides. By cross-priming, the host's immune system is awakened to the presence of cancer cells and an intrinsic anti-cancer vaccination effect against cancer is obtained.

Innate 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, but tumor cells One mechanism to avoid phagocytosis by innate immune cells is to upregulate CD47, a 'do not eat me' signal. Blocking the CD47-Sirpα axis between tumor cells and phagocytic cells. When used, it was shown to increase phagocytosis against tumor cells, proving its advantage as a target in cancer immunotherapy.In addition, CD47-based therapy induces innate and adaptive immune responses in an immunocompetent mouse model. It has been reported to promote anti-cancer immune effects by inducing connections between the liver (Liu , However, it is known that the anticancer activity of these Sirpα variants themselves is not as excellent as expected.

Accordingly, in order to improve CD47-mediated immunotherapy, the present inventors have made diligent efforts to express the Sirp protein, which can bind and antagonize human and mouse CD47, by fusing it with human ferritin heavy chain protein to form a multimer. Meanwhile, when this multimerized fusion protein is treated in combination with an immunogenic apoptosis inducing agent such as doxorubicin, the function of these Sirp proteins is significantly improved, inhibiting the evasion of cancer cells from immune cells and further increasing the death of cancer cells by immune cells. It was confirmed that it could be improved.

The present inventors have studied strategies for the secretion of immunogenic neoantigens and danger signals from dying cancer cells, and as a result, the combined treatment of the multimeric Sirp and immunogenic apoptosis inducer stimulates local inflammatory response and loads neoantigens. It was found that it triggers the creation of dendritic cells more strongly. Some dying cancer cells trigger a large-scale immune response, and this phenomenon is called ‘immunogenic cell death (ICD)’ (Kroemer et al . Annu. Rev. Immunol. 31: 51-72, 2013). The ICD communicates a combination of three distinct 'danger' signals limited in space and time:

1) “eat-me” signal, which involves translocation of calreticulin (CRT) from the endoplasmic reticulum (ER) to the cell surface; 2) “find-me” signal associated with activation of secretion of ATP; and 3) signals that promote antigen processing and presentation to T cells, involving extracellular secretion of nuclear high-mobility group box 1 (HMGB1) protein. These signals regulate a series of receptors expressed on the surface of dendritic cells to stimulate presentation of tumor neoantigens to T cells. Therefore, the present inventors hypothesized that an ICD inducer delivered together with a CD47 antagonist to the tumor microenvironment would trigger danger signals from dying cancer cells and initiate a cellular immune response.

As an ICD inducer for delivery together with multimeric Sirp, doxorubicin (dox), an anthracycline anticancer drug that induces the three properties of ICD in treated cancer cells, was selected, taking advantage of the metal ion-binding affinity of ferritin, a mediator of iron homeostasis. was used. The metal-ion binding affinity of ferritin allows metal-based drugs or metal-complex drugs to accumulate in the central cavity of ferritin. For this purpose, the present inventors prepared a doxorubicin preparation encapsulated in an FHSirpα nanocage by internalizing doxorubicin precomplexed with Cu(II) into the internal pores of the nanocage, and named it FHSirpα-Dox. Successful loading of doxorubicin into FHSirpα was confirmed by size-limited chromatography and the amount of captured doxorubicin was found to be 54 doxorubicin molecules per FHSirpα nanocage.

As a result of administering the FHSirpα-Dox to tumor model mice, strong synergistic anticancer activity was confirmed, reflecting the favorable tumor accumulation of a large amount of multimerized CD47 antagonist and ICD. Stimulation of local inflammatory responses and phagocytosis and maturation of innate immune cells in the immunosuppressive tumor microenvironment lead to effective delivery and presentation of immunogenic tumor neoantigens to T cells. This powerful immune response resulted in total tumor eradication and sustained anti-tumor immune response and, overall, proved to be a universal and effective approach to activating the host immune system against tumors.

In comparison, current cancer vaccines are limited in that they require continuous expression of the desired target tumor neoantigen 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 sustained expression of the desired target tumor neoantigen, optimization requirements, and economic costs of ex vivo manipulation, and the need for PD-1 antibodies. The same regulatory antibodies have the disadvantage that all activated or exhausted T cells, including anti-tumor and anti-autoimmune T cells, can be stimulated. However, the combined administration of the multimeric Sirp protein and immunogenic apoptosis inducer of the present invention activates both local and systemic anti-tumor specific immunity, resulting in its efficacy as an 'intrinsic anti-cancer vaccination'. Considering that it is a powerful immune stimulant that demonstrates durability and robust response, it is believed that the synergistic effect has broad potential and can be used to treat multiple types of cancer regardless of stage.

Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, the present invention is not limited to the examples and experimental examples disclosed below, but can be implemented in various different forms. The following examples and experimental examples ensure that the disclosure of the present invention is complete and are based on common knowledge. It is provided to fully inform those who have the scope of the invention.

Example 1: Preparation of ferritin nanocages

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

The present inventors have developed a polynucleotide encoding human ferritin heavy chain protein (hFTH) consisting of the amino acid sequence shown in SEQ ID NO: 1 (SEQ ID NO: 2); A polynucleotide encoding a linker peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 (SEQ ID NO: 4) and a polynucleotide encoding a Sirpα high-affinity variant consisting of the amino acid sequence shown in SEQ ID NO: 5 (SEQ ID NO: 6) Each was cloned by PCR or synthesis, ligated using restriction enzymes and ligase, and then cloned into the expression vector pT7-7 containing a His tag. In order to facilitate cloning of polynucleotides encoding each protein or peptide, 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. In addition, a restriction enzyme Cla I recognition site was added to the 3'-end of the polynucleotide encoding Sirpα.

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, E. coli BL21(DE3) treated with CaCl ₂ was transformed with the vectors prepared above using the heat shock method, and then cultured in a medium containing ampicillin to select cells transformed with the expression vector and showing ampicillin resistance. did. Transformed cells were cultured at 36°C until OD ₆₀₀ reached 0.6, then 1 mM IPTG was added to induce expression of the fusion protein, and the cells were further cultured at 20°C for 16 hours. After completing the culture, the cells were recovered and disrupted by ultrasonic waves, and the cell lysate was centrifuged at 12,000 g for 30 minutes to remove cell debris. Recombinant proteins were 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: pH 8.0, 50 mM mM sodium phosphate, 300 mM NaCl, 250 mM imidazole). To remove imidazole in the elution buffer, the buffer was replaced with PBS using a membrane filter (Amicon, 10K). The concentration of the obtained nanocage was measured using the Bradford protein analysis method. The nanocage prepared as above was named 'FHSirpα HV'.

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

The present inventors used the same method as Example 1-1 except that a polynucleotide (SEQ ID NO: 8) encoding the human Sirpα wild-type protein shown in SEQ ID NO: 7 rather than a Sirpα high-affinity variant was used, and the human ferritin heavy chain protein was added in the same manner as in Example 1-1. A fusion protein linked to the Sirpα wild-type protein and a ferritin heavy chain nanocage (hereinafter abbreviated as 'FH-hSirpα WT nanocage') using the same were prepared.

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

The present inventors added Sirpγ wild type to ferritin heavy chain protein in the same manner as Example 1-1, except that a polynucleotide (SEQ ID NO: 10) encoding the Sirpγ wild-type protein shown in SEQ ID NO: 9 was used instead of the Sirpα high-affinity variant. A protein-linked ferritin heavy chain nanocage (hereinafter abbreviated as 'FHSirpγ WT nanocage') was prepared.

1-4: Ferritin heavy chain protein and Sirp-γ variant 1 fusion protein

The present inventors have identified a polynucleotide encoding a Sirpγ variant (hereinafter abbreviated as 'Sirpγ V1', SEQ ID NO: 11), which has an amino acid mutation corresponding to the mutant amino acid of the Sirpα high-affinity variant in the Sirpγ wild-type protein, which is not a Sirpα high-affinity variant. A ferritin heavy chain nanocage (hereinafter abbreviated as 'FHSirpγ V1 nanocage'), in which the Sirpγ V1 protein is linked to the ferritin heavy chain protein, was prepared in the same manner as in Example 1-1, except that (SEQ ID NO: 12) was used. .

1-5: Ferritin heavy chain protein and Sirp-γ variant 2 fusion protein

The present inventors have identified a Sirpγ variant (hereinafter referred to as 'Sirpγ') having an amino acid mutation corresponding to the mutant amino acid of the Sirpα high-affinity variant, except that the 27th amino acid is not substituted (valine) in the Sirpγ wild-type protein, which is not a Sirpα high-affinity variant. A ferritin heavy chain nanocage (abbreviated as HV2', where the Sirpγ V2 protein is linked to the ferritin heavy chain protein in the same manner as in Example 1-1 except that a polynucleotide (SEQ ID NO: 14) encoding (SEQ ID NO: 13) was used. Hereinafter abbreviated as 'FHSirpγ V2 nanocage') was prepared.

Example 2: Preparation of doxorubicin loaded nanocages

The present inventors first reacted 1 mg/ml of doxorubicin with 1 mM copper ions (Cu ²⁺ ) at room temperature for 30 minutes to form a doxorubicin-copper ion complex. Then, the mixture was added to the FHSirpα HV solution (250 μg/ml) prepared in Example 1-1 and reacted at room temperature for 120 minutes. Free doxorubicin and copper ions were removed from the reaction product by chromatography using a PD-10 column. The loaded doxorubicin was measured using a fluorescence spectrometer (2103 EnVision™ Multilabel Plate Readers, PerkinElmer, USA) and then quantified by comparison with a standard curve. In order to confirm whether nanoparticles were formed in the prepared doxorubicin composite nanocage, the recovered The particle size of the nanocage was analyzed using a dynamic light scattering (DLS) analyzer (Malvern zetasizer nano ZS, UK), and the resulting nanocage was photographed using a transmission electron microscope.

As a result, as shown in Figure 1b, the doxorubicin complex nanocage of the present invention also appeared as a single peak on FPLC, and the absorbance measurement result at 480 nm for doxorubicin measurement also showed the same retention as the protein detection at 280 nm wavelength. By checking the time, it was indirectly confirmed that doxorubicin was successfully loaded inside the doxorubicin composite nanocage of the present invention. As shown in Figure 1c, the particle size analysis results confirmed that the nanoparticles were nanoparticles with a particle size of 10 to 100 nm (average 19.38ㅁ1.7 nm), and the transmission electron microscopy results also showed spherical nanoparticles as shown in Figure 1d. formation could be confirmed.

Example 3: Preparation of FcSirpα

The present inventors have provided a polynucleotide (SEQ ID NO: 90) encoding the Fc domain of human IgG1 shown in SEQ ID NO: 89, a polynucleotide (SEQ ID NO: 4) encoding a linker peptide consisting of the amino acid sequence shown in SEQ ID NO: 3, and a sequence A polynucleotide encoding a fusion protein to which a polynucleotide (SEQ ID NO: 6) encoding a Sirpα high-affinity variant consisting of the amino acid sequence indicated by number 5 is linked is cloned by PCR or synthesis, and then subjected to restriction enzymes and ligase. After linking using , it was cloned into the expression vector pT7-7 containing a His tag. In order to facilitate cloning of polynucleotides encoding each protein or peptide, a restriction enzyme Xho I recognition site was added between Fc and the linker peptide, and a restriction enzyme Hin d III recognition site was added between the linker peptide and Sirpα. In addition, a restriction enzyme Cla I recognition site was added to the 3'-end of the polynucleotide encoding Sirpα.

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, E. coli BL21(DE3) treated with CaCl ₂ was transformed with the vectors prepared above using the heat shock method, and then cultured in a medium containing ampicillin to select cells transformed with the expression vector and showing ampicillin resistance. did. Transformed cells were cultured at 36°C until OD ₆₀₀ reached 0.6, then 1 mM IPTG was added to induce expression of the fusion protein, and the cells were further cultured at 20°C for 16 hours. After completing the culture, the cells were recovered and disrupted by ultrasonic waves, and the cell lysate was centrifuged at 12,000 g for 30 minutes to remove cell debris. Recombinant proteins were 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: pH 8.0, 50 mM mM sodium phosphate, 300 mM NaCl, 250 mM imidazole). To remove imidazole in the elution buffer, the buffer was replaced with PBS using a membrane filter (Amicon, 10K). The concentration of the obtained fusion protein was measured using the Bradford protein analysis method. The fusion protein prepared as above was named 'FcSirpα'.

Experimental Example 1: FHSirpα-Dox in vivo Analysis of anticancer effect

1-1: Anticancer effect analysis using CT26.CL25 orthotopic tumor mice

The present inventors used a xenograft cancer model mouse to confirm whether the combination administration agent (FHSirpα-Dox) of Sirpα multimer and doxorubicin prepared according to an embodiment of the present invention actually exhibits the same anticancer effect in animal model experiments. The anticancer activity of FHSirpα-Dox prepared in Example 2 of the invention was investigated. Balb/c wild-type mice were used as experimental animals, and experiments on the experimental animals were conducted in accordance with the regulations of the KIST Animal Ethics Committee.

First, to measure the in vivo anticancer activity of various forms of Sirpα protein and doxorubicin, ^1X106 CT26.CL25 cancer cells expressing β-galactose were subcutaneously injected into the left back of 8-week-old Balb/c wild-type mice to induce cancer. , On the 7th day of cancer cell injection, each test substance (buffer, dox, wtFH-dox, mSirpα + dox, FHSirpα-dox) was injected intravenously a total of 5 times at 3-day intervals. At this time, the dosage of each substance is 1 mg/kg of doxorubicin (dox), 15 mg/kg of wtFH-dox (equivalent to 1 mg/kg in terms of doxorubicin content), and 9.5 mg/kg of mSirpα + 1 mg/kg of dox. , and FHSirpα-dox was 28 mg/kg (corresponding to 1 mg/kg in doxorubicin content). Then, the length (L) and width (W) of the cancer cells were measured using a caliper at 3-day intervals until the 25th day of cancer cell injection, and then calculated using the following formula (Figure 2a), and on the 25th day, the experimental animals After photographing the tumor tissue area, the tumor tissue was extracted and the weight of the tumor tissue was measured (Figures 2b to 2d):

Cancer cell volume (V[mm ³ ])=(L[mm])x(W[mm]) ² x0.5

As a result, as shown in Figures 2b and 2c, the size of the tumor in the control group (only buffer injection) exceeded 1,000 mm ³ , and the effect of the group treated with doxorubicin alone was also minimal. In addition, when doxorubicin was loaded onto wild-type ferritin (wtFH-dox) and when wild-type Sirpα and doxorubicin were administered together (mSirpα + dox), an anticancer effect was observed, but it was not at a level that sufficiently inhibited the growth of cancer cells. On the other hand, in the case of the combined administration of Sirpα multimer and doxorubicin (FHSirpα-dox) according to an embodiment of the present invention, the growth of cancer cells was significantly inhibited to the extent that the volume of cancer cells was about 200 mm ³ even after 25 days. Furthermore, the combined administration of Sirpα multimer and doxorubicin (FHSirpα-dox) according to an embodiment of the present invention almost completely inhibited the growth of cancer cells, showing such an effect that cancer cells could not be distinguished with the naked eye.

The above results were slightly different from the results of in vitro experiments. When doxorubicin was administered alone, recombinant Sirpα, which induced phagocytosis of macrophages in in vitro tests, was administered alone, or doxorubicin and monomeric recombinant Sirpα were administered in combination to animals. Considering that the anticancer effect was not very significant in the model experiment, this can be said to be a very groundbreaking result.

In addition, as a result of measuring the size of the tumor tissue extracted after sacrificing the experimental animals 25 days after cancer cell injection, as shown in Figure 2d, the combined administration of the Sirpα multimer of the present invention and doxorubicin showed a significant cancer cell reduction effect. Inhibition of cancer cell growth was limited in the combined administration of doxorubicin (wtFH-dox), a combination of doxorubicin and doxorubicin encapsulated in regular ferritin without Sirpα multimers, the combined administration of doxorubicin and monomeric recombinant Sirpα, and the administration of doxorubicin alone.

1-2: Immunogenic cell death analysis

The present inventors performed flow cytometric analysis on cancer cells to confirm whether immunogenic cell death is induced by the combined administration of Sirpα multimer and doxorubicin according to an embodiment of the present invention.

Specifically, the present inventors inoculated CT26.CL25 cancer cell 1ㅧ10 ⁶ cells into 8-week-old Balb/c mice (day 0), and then administered experimental substances (buffer, dox, wtFH-dox, mSirpα +) to each group from day 7. dox, FHSirpα-dox) was injected intravenously a total of 5 times at 3-day intervals. At this time, the dosage of each substance is 1 mg/kg of doxorubicin (dox), 15 mg/kg of wtFH-dox (equivalent to 1 mg/kg in terms of doxorubicin content), and 9.5 mg/kg of mSirpα + 1 mg/kg of dox. , and FHSirpα-dox was 28 mg/kg (corresponding to 1 mg/kg in doxorubicin content). On the 25th day after cancer cell injection, the tumor tissue was excreted and converted into single cells using DNase and collagenase, and dendritic cells and macrophages in the tumor microenvironment were treated using anti-CD11c antibody and anti-F4/80 antibody. The amount present was examined by FACS analysis (Figures 3a and 3b).

As a result, as shown in Figures 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 compared to the control group. This means that FHSirpα-dox according to an embodiment of the present invention induces an innate immune response through mobilization of immune cells against cancer cells.

1-3: Antigen-induced immune response analysis

To confirm whether the immune response occurred specifically for cancer cells, the present inventors examined CT26 in the mouse spleen and tumor-draining lymph nodes by the combined administration of FHSirpα multimer and doxorubicin (FHSirpα-dox) according to an embodiment of the present invention. Cellular immune responses to CL25 cancer cells were investigated. That is, the level of interferon-gamma (INF-γ) in T cells specific for β-galactosidase, an antigen of CT26.CL25, was quantified using ELISA (R&D Systems, Inc, USA) analysis. Three mice per group treated in the same manner as in Experimental Example 1-1 were selected, the spleen and tumor-drained lymph node (TDLN) were removed from each mouse, and the tissues were placed in a sterilized Petri dish. Then, the spleen and tumor-draining lymph nodes were ground using a cell strainer to separate the cells from the tissue capsule. All contents of the Petri dish were transferred to a 15 ml tube, filled with RPMI 1640 medium, centrifuged at 500G for 10 minutes, and the supernatant was removed. Red blood cells were hemolyzed using red blood cell lysis buffer (Sigma-Aldrich, Germany) in the pellet. . The cells contained in the tube were washed with PBS and suspended in RPMI 1640 medium to separate tumor-drained lymph node (TDLN) cells and splenocytes. Separated TDLN cells and spleen cells were seeded in a 24-well plate at 1ㅧ10 ⁶ cells/500 μl and 1ㅧ10 ⁷ cells/ml, respectively, and β-gal peptide (TPHPARIGL, SEQ ID NO: 87, amino acid residue of β-gal) Contains the naturally engineered H-2 Ld restricted epitope encompassing 876-884), AH1 (SPSYVYHQF, SEQ ID NO: 88, an epitope from the gp70 protein, an endogenous antigen of CT26.CL25, and contains the CTL determinant from CT26 ), treatment with P1A peptide (negative control) at a concentration of 5 μg/ml for 24 hours promoted the activation of CD8 ⁺ T cells that secrete IFN-γ. Afterwards, the supernatant was separated and the level of interferon-gamma (INF-γ) was quantified using ELISA (R&D Systems, Inc, USA) analysis.

As a result, as shown in Figures 4a and 4b, when the tumor-draining lymph node cells and spleen immune cells isolated from animals administered FHSirpα-dox according to an embodiment of the present invention are treated with β-gal peptide and AH1 peptide , it was confirmed that the expression of INF-γ significantly increased. In particular, in the case of the FHSirpα nanocage loaded with doxorubicin according to an embodiment of the present invention, the expression level of interferon gamma in spleen cells was recorded at more than 100 pg/ml, showing a very high degree of immune activation against cancer cells. Other agents, such as co-administration of doxorubicin and recombinant Sirpα, showed little or no effect. In other words, in the case of FHSirpα-dox, it effectively delivers β-galactosidase protein to macrophages and dendritic cells to activate the function of antigen-presenting cells, and ultimately effectively stimulates cytotoxic T cells to produce a specific immune response to CT26.CL26 cancer cells. It was confirmed that it was effectively induced.

1-4: Immunohistochemical analysis

In order to confirm whether the results of Experimental Example 1-3 were an effect of the recruitment of immune cells to cancer cell tissue, the present inventors sliced the tumor tissue extracted in Experimental Example 1-3 and identified CD8, a T cell marker. Immunohistochemical analysis was performed.

Specifically, the tumor tissue extracted from the experimental animal administered FHSirpα-dox prepared in Experimental Example 1-3 was fixed in 10% neutral formalin solution, a paraffin block was prepared, and this was prepared into a 4 μm thick slice, and then adjusted to pH 6.0 Antigen retrieval was performed by boiling in an aqueous citric acid solution, followed by washing three times with TBST (Tris-buffered saline, Tween-20) and blocking using Renaissance Ab diluent (PD905, Biocare Medical). Performed at room temperature for 10 minutes, reacted with anti-CD8 antibody (BD Bioscience, USA), washed three times, reacted with anti-rat secondary antibody (MP-7445-15, Vector Laboratories, USA), and then reacted with anti-CD8 antibody (BD Bioscience, USA). After washing sequentially and performing tyramine signal amplification (TSA) using a tyramine-conjugated fluorophore (NEL794001KT, PerkinElmer, USA), washing was performed three times, and non-specific triamine To remove the fluorophore binding, it was boiled in an aqueous citric acid solution, stained with DAPI, and photographed using a PerkinElmer Vectra platform.

As a result, as shown in Figure 5, CD8 ⁺ T cells within the tumor tissue were positively stained. This proves that CD8 ⁺ T cells have infiltrated into the tumor tissue, suggesting that the above anticancer effect is due to the synergistic effect of recruitment of immune cells according to CD47 masking of doxorubicin and Sirpα.

1-5: Anticancer effect upon intratumoral injection

The present inventors investigated whether the combined administration of Sirpα multimer and doxorubicin (FHSirpα-Dox) according to an embodiment of the present invention shows equivalent anticancer effects when administered intratumorally in addition to intravenous injection.

Specifically, cancer was induced in 8-week-old Balb/c wild-type mice by subcutaneously inoculating CT26.CL25 cancer cells with 1ㅧ10 ⁶ cells on the left back, and each test substance (buffer, wtFH-dox, and FHSirpα) was administered on the 7th day after cancer cell injection. -dox) was administered intratumorally once. At this time, the dosage of each substance was 15 mg/kg of wtFH-dox (corresponding to 1 mg/kg in doxorubicin content), and 28 mg/kg of FHSirpα-dox (corresponding to 1 mg/kg in doxorubicin content). ) was. Then, the volume of cancer cells was measured at 3-day intervals until the 25th day of cancer cell injection as described in Experimental Example 1-1, and on the 25th day, tumor tissues from the experimental animals were removed and the weight of the tumor tissues was measured.

As a result, as shown in Figure 6a, when doxorubicin was loaded on wild-type ferritin, the tumor growth rate was lower than the control group, but the tumor tissue still grew. On the other hand, in the case of the combined administration of Sirpα multimer and doxorubicin (FHSirpα-dox) according to an embodiment of the present invention, cancer cells hardly grew even after 25 days. In addition, as a result of measuring the weight of the extracted tumor tissue, as shown in Figure 6b, in the case of wtFH-dox, the weight of the tumor tissue was reduced to less than half compared to the control group, which exceeded 1.0 g, to about 0.5 g, whereas the weight of the tumor tissue of the present invention was reduced to about 0.5 g. In the case of FHSirpα-dox according to one example, it was safe to say that there was almost no tumor. This demonstrates that the combined administration of Sirpα multimer and doxorubicin according to an embodiment of the present invention has a more excellent anticancer effect when administered intratumorally.

Experimental Example 2: Analysis of the anticancer memory effect of FHSirpα-Dox

From the results of Experimental Examples 1-3, the present inventors concluded that since the action of the FHSirpα nanocage according to an embodiment of the present invention is an effect by recruiting immune cells, there will be a memory effect according to the memory of immune cells. Assuming that the tumor tissue extracted after administration of FHSirpα-dox according to an embodiment of the present invention was transplanted to the other back of the same animal without being sacrificed, the growth of cancer at the transplanted location was analyzed over time, and The number of surviving mice was counted.

As a result, as shown in Figures 7a and 7b, in animals transplanted with tumor tissue extracted from the FHSirpα-dox administration group according to an embodiment of the present invention, not a single mouse had cancer cells growing until 28 days had passed. This was not observed, and mice transplanted with tumor tissue extracted from groups administered other substances showed recurrence of cancer over time, albeit to varying degrees. In addition, as a result of analyzing the survival rate after 80 days for the same experimental group, as shown in Figure 7c, in the FHSirpα-dox administered group according to an embodiment of the present invention, none of the experimental animals died until 80 days had elapsed. In the case of FHSirpα, the survival rate after 80 days was 80%, which was the second most effective treatment after FHSirpα-Dox. When treated with doxorubicin alone or recombinant Sirpα alone, the survival rate was 50% after 80 days.

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 that can suppress recurrence after cancer treatment. Therefore, the nanocage according to an embodiment of the present invention can be very effective in not only treating cancer but also suppressing recurrence.

Experimental Example 3: Analysis of anticancer effect of FHSirpα-Dox on various tumors

3-1: Analysis of anticancer effect on CT26 cells

In order to confirm whether the combined administration of Sirpα multimer and doxorubicin according to an embodiment of the present invention exhibits an anticancer effect on other types of cancer cells, the present inventors used CT26 cells, which are general colon cancer cells that do not express β-galatosidae. The anticancer effect on existing cancer model animals was analyzed.

Specifically, cancer was induced in 8-week-old Balb/c wild-type mice by subcutaneously inoculating CT26 cancer cell 1ㅧ10 ⁶ cells on the left back, and each test substance (buffer, dox, wtFH-dox, mSirpα + dox) was administered on the 7th day after cancer cell injection. , FHSirpα-dox) was injected intravenously a total of 5 times at 3-day intervals. At this time, the dosage of each substance is 1 mg/kg of doxorubicin (dox), 15 mg/kg of wtFH-dox (equivalent to 1 mg/kg in terms of doxorubicin content), and 9.5 mg/kg of mSirpα + 1 mg/kg of dox. , and FHSirpα-dox was 28 mg/kg (corresponding to 1 mg/kg in doxorubicin content). Then, the volume of the cancer cells was measured at 3-day intervals until the 25th day after cancer cell injection, as described in Experimental Example 1-1 above, and on the 25th day, the tumor tissues of the experimental animals were extracted and weighed.

As a result, as shown in Figures 8a and 8b, in the case of dox, wtFH-dox, and mSirpα + dox, the degree of inhibition of the growth of cancer cells was minimal compared to the control group, whereas in the case of FHSirpα-dox according to an embodiment of the present invention, cancer cells were inhibited. I was able to confirm that it had barely grown. As a result of measuring the weight of the extracted cancer cells, the weight of the tumor tissue was not significantly lower in the case of dox and mSirpα + dox compared to the control group, and in the case of wtFH-dox, the weight of the tumor tissue was significantly lower than the control group (P < 0.05). The size decreased, but the degree was weak. On the other hand, in the case of FHSirpα-dox according to an embodiment of the present invention, it was safe to say that there was almost no tumor tissue.

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

The present inventors analyzed the anticancer activity of B16F10-Ova cells, a type of melanoma that expresses ovalbumin.

Specifically, the present inventors specifically induced cancer in 8-week-old Balb/c wild-type mice by subcutaneously inoculating B16F10-Ova cancer cells 1ㅧ10 ⁶ cells on the left back, and administered each test substance (buffer, dox) on the 7th day after cancer cell injection. , wtFH-dox, mSirpα + dox, FHSirpα-dox) were injected intravenously a total of three times at three-day intervals. At this time, the dosage of each substance is 1 mg/kg of doxorubicin (dox), 15 mg/kg of wtFH-dox (equivalent to 1 mg/kg in terms of doxorubicin content), and 9.5 mg/kg of mSirpα + 1 mg/kg of dox. , and FHSirpα-dox was 28 mg/kg (corresponding to 1 mg/kg in doxorubicin content). Then, the volume of the cancer cells was measured at 3-day intervals until the 25th day after cancer cell injection, as described in Experimental Example 1-1 above, and on the 25th day, the tumor tissues of the experimental animals were extracted and weighed.

As a result, as shown in Figures 9a and 9b, in the case of dox, wtFH-dox, and mSirpα + dox, the degree of inhibition of the growth of cancer cells was minimal compared to the control group, whereas in the case of FHSirpα-dox according to an embodiment of the present invention, cancer cells were inhibited. I was able to confirm that it had barely grown. As a result of measuring the weight of the extracted cancer cells, the weight of the tumor tissue was lower in the case of dox, wtFH-dox, and mSirpα + dox compared to the control group, but it was not significant. On the other hand, in the case of FHSirpα-dox according to an embodiment of the present invention, it was safe to say that there was almost no tumor tissue.

3-3: Cross-priming ability analysis

The present inventors investigated whether the combination of Sirpα multimer and doxorubicin according to an embodiment of the present invention had the cross-priming ability of dendritic cells. Cross-priming is when a specific antigen-presenting cell ingests an extracellular antigen, processes it, and presents it together with MHC class 1 molecules to CD8 ⁺ T cells (cytotoxic T cells). presentation), refers to the phenomenon in which naïve CD8 ⁺ T cells are stimulated and differentiated into cytotoxic CD8 ⁺ T cells.

For this purpose, cancer was induced in 8-week-old Balb/c wild-type mice by subcutaneously inoculating B16F10-Ova cancer cells 1ㅧ10 ⁶ cells on the left back. On the 7th day of cancer cell inoculation, buffer solution was administered as a control and FHSirpα-dox 28 mg as an experimental group. /kg (1 mg/kg as doxorubicin) was injected intravenously a total of 3 times at 3-day intervals. Finally, two days after the intravenous injection, the cancer tissue was extracted, converted into single cells using DNase and collagenase, and then CD11c positive cells (dendritic cells) were isolated using the MACS (magnetic-activated cell sorting) method. Afterwards, co-culture was performed for 3 days with OT-1 cells, a type of naive CD8 ⁺ T cells, and then the culture supernatant was obtained and the amount of INF-γ was measured by ELISA analysis.

As a result, as shown in Figure 10, in the case of the FHSirpα-dox treatment group according to an embodiment of the present invention, more than 100 pg/ml of INF-γ was secreted, whereas in the control group, INF-γ expression was extremely minimal. This means that the dendritic cells of the FHSirpα-dox administered group according to an embodiment of the present invention have the cross-priming ability to stimulate uncontacted CD8 ⁺ T cells by presenting cancer cell antigens together with MHC type 1 molecules to uncontacted CD8 ⁺ T cells. This suggests that

Subsequently, the present inventors performed the following additional analysis to confirm whether the above phenomenon was a true cross-priming ability.

Specifically, cancer was induced in 8-week-old Balb/c wild-type mice by subcutaneously inoculating B16F10-Ova cancer cell 1ㅧ10 ⁶ cells on the left back, and each test substance (buffer, dox, FHSirpα and FHSirpα-dox) was administered on the 7th day of cancer cell inoculation. ) was injected intravenously twice, at 3-day intervals. The dosage of each test substance used at this time was 1 mg/kg for doxorubicin (dox), 28 mg/kg for FHSirpα, and 28 mg/kg for FHSirpPα-dox (1 mg/kg based on dox). Finally, 10 days after intravenous administration, OT-1 T cells stained with CFSE (carbolxyfluorescein succinimidyl ester), a cell tracking agent, were injected intravenously, and after 3 days, tumor draining lymph nodes (TDLN) were removed and single After cellization, the degree of cell proliferation of OT-1 T cells was confirmed through CFSE proliferation analysis through FACS analysis, and the cross-priming ability of each group was analyzed.

As a result, as shown in Figures 11a and 11b, the injected CFSE-stained OT-1 T cells proliferated more effectively in the case of FHSirpα-dox according to an embodiment of the present invention compared to the buffer, dox, and FHSirpα groups. A trend was confirmed. Specifically, in the case of FHSirpα-dox, the proportion of proliferated 7th generation OT-1 T cells was 15%, which was significantly increased compared to the other groups.

Experimental Example 4: Anticancer effect of FcSirpα + Mtx

The present inventors investigated the anticancer effect of the FcSirpα fusion protein prepared in Example 3 and the anticancer drug mitoxantrone in combination to determine whether the combined effect of Sirpα and the anticancer drug can be implemented on other platforms.

4-1: Analysis of phagocytosis by FcSirpα

First, the present inventors analyzed the phagocytosis of cancer cells using bone marrow-derived macrophages (BMDM) to investigate the effect of FcSirpα on the phagocytosis of macrophages against cancer cells. For this purpose, specifically prepared BMDMs were stained with 1 μM CellTracker Green CMFDA (Thermo Fisher Scientific, USA). 20x10 ⁴ cells of each stained macrophage were seeded in a 35 mm confocal dish with 2 ml RPMI medium. Bone marrow-derived macrophages prepared in RPMI medium and the HT29 cancer cell line stained with 120 ng/ml pH rodo SE (Thermo Fisher Scientific, USA) were incubated with control or FcSirpα 5 prepared in Example 1 at about 37°C for 2 hours. Co-cultured with μM or 400 nM of FHSirpα prepared in Example 1. At this time, FcSirpα 5 μM and FHSirpα 400 nM contain the same amount of Sirpα. After 2 hours of co-culture, the degree of phagocytosis of cancer cells was analyzed using a fluorescence microscope (Figure 12a, red: phagocytosed cancer cells, green: bone marrow-derived macrophages), and quantified (Figure 12b). As a result, as seen in Figures 12a and 12b, the phagocytic ability of macrophages to cancer cells was significantly increased by FcSirpα and FHSirpα, and among these, FcSirpα was confirmed to have the best phagocytic ability.

Next, in order to determine at what concentration the phagocytic activity of FcSirpα appears, the present inventors analyzed the phagocytic activity on cancer cells using flow cytometry at different concentrations of FcSirpα. For this purpose, specifically prepared BMDMs were stained with 1 μM CellTracker Green CMFDA (Thermo Fisher Scientific, USA). 20x10 ⁴ cells of each stained macrophage were seeded in a 35 mm Peptri dish with 2 ml RPMI medium. Bone marrow-derived macrophages prepared in RPMI medium and the HT29 cancer cell line stained with 1 μM CellTracker Deep redd (Thermo Fisher Scientific, USA) were incubated with FcSirpα prepared in Control or Example 1 or Example for 2 hours at about 37°C. It was co-cultured with FHSirpα prepared in 1. At this time, FcSirpα 5 μM and FHSirpα 400 nM contain the same amount of Sirpα. After 2 hours of co-culture, the degree of phagocytosis of cancer cells was analyzed using a flow cytometer. As shown in Figure 12c, the phagocytosis of macrophages was determined by FcSirpα 50 nM, FcSirpα 500 nM, FcSirpα 5 μM, and FHSirpα 400 nM. Cancer cell phagocytic ability was significantly increased, and in particular, FcSirpα was confirmed to have excellent phagocytic ability starting from a concentration of 50 nM.

4-2: FcSirpα + by mitoxantrone in vivo Anti-cancer effect analysis

Based on the above experimental results, the present inventors investigated the synergistic effect of combined use of FcSirpα and mitoxantrone, another immunogenic apoptosis inducer.

For this purpose, cancer was induced in C57BL/6 wild type mice by subcutaneously injecting ⁵ μg or FcSirpa was administered by intratumoral injection at a dose of 400 μg. In the case of the FcSirpα + MTX group, Mitoxantrone was administered by intratumoral injection at a dose of 400 μg FcSirpα 4 hours later. After drug administration, the cancer size was measured at 3-day intervals (Figure 13a), and the survival rate up to 21 days after cancer cell injection was recorded (Figure 13b). On the 21st day after cancer cell injection, the experimental mice were sacrificed, the cancer cells were extracted, and the weight was calculated. was measured (Figure 13c). As shown in Figures 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

Based on the results of Experimental Example 4-2, the present inventors evaluated whether the adaptive immune response was also enhanced when FcSirpα + MTX was administered in combination by measuring the degree of infiltration of CD8 ⁺ T cells into the tumor microenvironment. For this purpose, cancer was specifically induced in C57BL/6 wild type mice by subcutaneously injecting ⁵ It was administered by intratumoral injection at a dose of μg. In the case of the FcSirpα group, 10 μg of Mitoxantrone was administered intratumorally, and 4 hours later, FcSirpa was administered by intratumoral injection at a dose of 400 μg. Twenty-one days after cancer cell injection, mice were sacrificed, cancer tissues were extracted, single cells were made, and the degree of CD8 ⁺ T cell infiltration within the cancer tissues was compared and analyzed through flow cytometry after staining with anti-CD8 antibody. As a result, as seen in Figure 14, a significant increase in CD8 ⁺ T cells was confirmed in the FcSirpα + MTX group compared to the other groups. This shows that the multimeric Sirpα fusion protein according to an embodiment of the present invention exhibits surprising anticancer activity by enhancing not only the innate immune response but also the adaptive immune response when administered in combination with an immunogenic apoptosis inducing agent.

The above experimental results show that the combined administration of signal regulatory proteins such as Sirpα and immunogenic apoptosis inducers such as doxorubicin or mitoxantrone strengthens the individual's immune response, especially the innate immune response, greatly promoting immunological death of cancer cells. This shows that Sirp multimerization plays a very important role in this anticancer activity. In this way, if a combination drug is developed that multimerizes the Sirp protein using the multimerization domain and combines it with an anti-inflammatory compound that induces immunogenic cell death, such as doxorubicin, it can effectively treat incurable cancers that were previously difficult to treat. It is expected that the development of a treatment will be possible.

The present invention has been described with reference to examples, but these are merely examples, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true scope of technical protection of the present invention should be determined by the technical spirit of the attached patent claims.

<110> Korea Institute of Science and Technology <120> A novel pharmaceutical composition for treating cancer <130> PD18-5703-D2 <150> KR 10-2017-0126259 <151> 2017-09-28 <160> 90 <170 > KoPatentIn 3.0 <210> 1 <211> 182 <212> PRT <213> Homo sapiens <400> 1 Thr Thr Ala Ser Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp Ser 1 5 10 15 Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu Glu Leu Tyr Ala Ser Tyr 20 25 30 Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu 35 40 45 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu 50 55 60 His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile 65 70 75 80 Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser Gly 85 90 95 Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val Asn Gln 100 105 110 Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His 115 120 125 Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys Ala 130 135 140 Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly Ala 145 150 155 160 Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly 165 170 175 Asp Ser Asp Asn Glu Ser 180 <210> 2 <211> 546 <212> DNA <213> Homo sapiens <400> 2 acgaccgcgt ccacctcgca ggtgcgccag aactaccacc aggactcaga ggccgccatc 60 aaccgccaga tcaacctgga gctctacgcc tcctacgttt acctgtccat gtcttactac 120 tttgaccgcg atga tgtggc tttgaagaac tttgccaaat actttcttca ccaatctcat 180 gaggagaggg aacatgctga gaaactgatg aagctgcaga accaacgagg tggccgaatc 240 ttccttcagg atatcaagaa accagactgt gatgactggg agagcgggct gaatgcaatg 300 gagtgtgcat tacatttgga aaaaaatgtg aatcagtcac tactggaact gcacaaactg 360 gccactgaca aaaatgaccc ccatttgtgt gacttcattg agacacatta cctgaatgag 420 caggtgaaag ccatcaaaga attgggtgac cacgtgacca acttgcgcaa gatgggagcg 480 cccgaatct g gcttggcgga atatctcttt gacaagcaca ccctggggaga cagtgataat 540 gaaagc 546 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> < 223> linker peptide <400> 3 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 <210> 4 <211> 93 <212> DNA <213> Artificial Sequence <220> <223 > polynucleotide encoding linker peptide <400> 4 ggcagctctg gtggcagcgg tagctctggc ggtagcggcg gtggcgatga agcggacggt 60 agccgcggct ctcagaaagc gggtgtggat gaa 93 <210> 5 <211> 118 <212> PRT <213> Artificial Sequence <22 0> <223> SiRP alpha variant <400> 5 Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Leu Val Ala 1 5 10 15 Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ile Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro 115 <210> 6 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding SiRP alpha variant <400> 6 gaagaggagc tgcagatcat ccagcctgac aagtccgtgc tggtcgctgc tggtgaaact 60 gccactctgc gttgtacgat taccagcctg ttcccggtgg gtccaatc ca gtggttccgt 120 ggtgctggtc cgggtcgtgt tctgatctac aaccagcgtc aaggtccgtt cccgcgtgta 180 actaccgtta gcgataccac gaagcgtaac aacatggact tttccatccg cattggcaat 240 attaccccgg ccgacgcggg cacctactat tgcatcaaat ttcgcaaagg ctccccggat 300 gatgtagaat ttaaatctgg cgcaggcacc gaactgtctg ttcgcgcaaa accgtaa 357 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> human wild type SIRP alpha <400> 7 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala 1 5 10 15 Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro 115 <210> 8 <211> 357 < 212> DNA <213> Artificial Sequence <220> <223> human wild type SIRP alpha <400> 8 gaggaggaat tacaggtcat tcaaccagat aaatcggtct tagtagcagc cggagagaca 60 gctacattga gatgtacggc gacaagcctt attcccgtgg ggccgatcca atggtttcgc 120 gg ggcaggcc ccggaagaga attgatttac aaccagaagg agggtcattt ccctcgcgtg 180 acgacggtca gcgacttaac taagcgtaat aacatggatt tttcaataag aataggcaat 240 ataactccgg ccgacgcagg gacgtactac tgtgttaaat tccggaaggg atctccggat 300 gatgtcgagt tcaaatctgg ggcgggtaca gaattgagcg ttcgggcaaa gccctaa 357 <210> 9 <211> 119 <212> PRT <213> Artificial Sequence < 220> <223> wild type SIRP gamma <400> 9 Glu Glu Glu Leu Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr 1 5 10 15 Val Gly Lys Thr Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro 20 25 30 Val Gly Pro Val Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser 65 70 75 80 Ile Thr Pro Ala Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Glu Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met 100 105 110 Ala Leu Gly Ala Lys Pro Ser 115 <210> 10 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> wild type SIRP gamma <400> 10 gaggaagaat tgcaaatgat ccagccggaa aaattattac tggttaccgt gggaaaaacg 60 gcgacccttc attgcacagt cacgtccctg ttgccggtag gtccagtttt gtggttccgg 120 gg ggttggac cagggcgtga actgatctat aatcaaaagg aaggtcattt tccgcgcgtg 180 accacagtga gcgatttgac taaacggaac aatatggact tctcgatccg catttctagt 240 attacaccgg cggacgttgg cacttattat tgcgtcaagt tccgcaaagg aagtcctgag 300 aacgtagagt tcaagtccgg tcctggcact gagatggctt tgggtgctaa accc 354 <210> 11 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SIRP gamma variant 1 (V1 ) <400> 11 Glu Glu Glu Leu Gln Ile Ile Gln Pro Glu Lys Leu Leu Leu Val Thr 1 5 10 15 Val Gly Lys Thr Ala Thr Leu His Cys Thr Ile Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Val Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser 65 70 75 80 Ile Thr Pro Ala Asp Val Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Glu Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met 100 105 110 Ala Leu Gly Ala Lys Pro 115 <210> 12 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding SIRP gamma variant 1 <400> 12 gaagaggagc tgcagatcat ccagcctgac aagtccgtgc tggtcgctgc tggtgaaact 60 gccactctgc gttgtacgat taccagcctg ttcccggtgg gtccaatcca gtggtt ccgt 120 ggtgctggtc cgggtcgtgt tctgatctac aaccagcgtc aaggtccgtt cccgcgtgta 180 actaccgtta gcgataccac gaagcgtaac aacatggact tttccatccg cattggcaat 240 attaccccgg ccgacgcggg cacctactat tgcatcaaat ttcgcaaagg ctccccggat 300 gatgtagaat ttaaatctgg cgcaggcacc gaactgtctg ttcgcgcaaa accg 354 <210> 13 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SI RP gamma variant 2 (V2) <400> 13 Glu Glu Glu Leu Gln Ile Ile Gln Pro Glu Lys Leu Leu Leu Val Thr 1 5 10 15 Val Gly Lys Thr Ala Thr Leu His Cys Thr Val Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Val Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser 65 70 75 80 Ile Thr Pro Ala Asp Val Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Glu Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met 100 105 110 Ala Leu Gly Ala Lys Pro 115 <210> 14 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding SIRP gamma variant 2 <400> 14 gaagaggaat tacaaatcat acaacctgaa aagctgttat tggtcaccgt aggcaaaacc 60 gctactctgc actgcactgt gacgtccctt tttcctgttg gtcctgtctt atggtttcg t 120 ggagtcggtc cgggtcgggt tcttatctat aaccagcggc aaggaccatt cccacgggtt 180 accacggttt cggacacaac gaaacgcaat aacatggatt tttccattcg gatttcaagc 240 atcactccgg ccgacgttgg aacttattac tgcataaagt ttagaaaggg atctccggag 300 aacgtagaat ttaagtctgg tccaggtact gagatggccc ttggagcgaa gccg 354 <210> 15 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Si RP alpha variant <400> 15 Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Ile Ser 50 55 60 Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro 115 <210> 16 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha D1 domain <400> 16 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro 115 <210> 17 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 17 Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Ile Ser 50 55 60 Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 18 <211> 119 <212> PRT <213> Artificial Sequence <220> < 223> SiRP alpha variant <400> 18 Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr 35 40 45 Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser 50 55 60 Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu 65 70 75 80 Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys 85 90 95 Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys 100 105 110 Tyr Arg Val Val Ser Thr Arg 115 <210> 19 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 19 Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Ile Ser 50 55 60 Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 20 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 20 Trp Lys Phe Lys Gly Arg Asp Ile Tyr 35 40 45 Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser 50 55 60 Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu 65 70 75 80 Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys 85 90 95 Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys 100 105 110 Tyr Arg Val Val Ser Trp Ser Thr Arg 115 120 <210> 21 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 21 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser Thr Arg 115 120 <210> 22 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 22 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser Thr Arg 115 120 <210> 23 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 23 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Arg Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 24 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400 > 24 Glu Glu Glu Val Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Gly Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 25 <211 > 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 25 Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Val Ile Leu His Cys Thr Ile Thr Ser Leu Thr Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Leu Leu 35 40 45 Ile Tyr Asn Gln Arg Glu Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Leu Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 26 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 26 Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Ser Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Gly Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Leu Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 27 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 27 Glu Glu Glu Ile Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Val Ile Ile His Cys Thr Val Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Arg Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Gly Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Val Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 28 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 28 Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ile Ile Leu His Cys Thr Val Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Glu Gly Arg Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Gly Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Leu Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 29 <211> 118 <212 > PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 29 Glu Glu Glu Val Gln Leu Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Glu Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Gly Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 30 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 30 Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Leu Val Ala 1 5 10 15 Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ile Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro Ser 115 <210> 31 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 31 Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Leu Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 32 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 32 Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Lys Gln Gly Pro Phe Pro Arg Val Thr Thr Ile Ser 50 55 60 Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 33 <211> 118 <212> PRT <213> Artificial Sequence <220> < 223> SiRP alpha variant <400> 33 Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Thr Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Gly Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 34 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 34 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala 1 5 10 15 Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro Ser 115 <210> 35 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 35 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 36 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 36 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu Leu Cys Thr Val Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 37 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 37 Glu Glu Gly Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ala Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro Ser 115 <210> 38 <211> 119 < 212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 38 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Phe Val Leu Val Ala 1 5 10 15 Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro Ser 115 <210> 39 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 39 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala 1 5 10 15 Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Pro Ile Arg Ile Gly Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro Ser 115 <210> 40 <211> 118 <212> PRT < 213> Artificial Sequence <220> <223> SiRP alpha variant <400> 40 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Gly Lys Pro Ser 115 <210> 41 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 41 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala 1 5 10 15 Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 42 <211> 119 <212> PRT <213> Artificial Sequence <220 > <223> SiRP alpha variant <400> 42 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala 1 5 10 15 Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro Ser 115 <210> 43 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 43 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 44 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 44 Glu Glu Glu Trp Phe Arg Gly Ala Gly Pro Gly Arg Xaa Leu 35 40 45 Ile Tyr Asn Gln Xaa 70 75 80 Ile Thr 210> 45 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 45 Glu Glu Glu Gly Glu Ser Xaa Ile Leu His Cys Thr Thr Val Ser 50 55 60 Glu Glu 46 Glu Glu Glu Gly Pro Ala Arg Xaa Leu 35 40 45 Ile Tyr Asn Gln Xaa Xaa Ala Asp Ala Gly Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu 119 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 47 Glu Glu Gly His Cys Thr Asp Ala Gly Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro Ser 115 <210> 48 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 48 Glu Glu Glu Xaa Gln Xaa Ile Gln Pro Asp Lys Phe Val Leu Val Ala 1 5 10 15 Ala Gly Glu Thr Leu 35 40 45 Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys 85 90 95 Gly Ser Pro Asp Asp Val Glu PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 49 Glu Glu Glu Thr Ser Leu Xaa Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Arg Asn Asn Met Asp Phe Pro Ile Arg Ile Gly Xaa 65 70 75 80 Ile Thr <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 50 Glu Glu Glu Xaa Ile Leu His Cys Thr 50 55 60 Glu Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Gly Lys Pro Ser 115 <210> 51 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 51 Glu Glu Glu Arg Xaa Leu 35 40 45 Ile Tyr Asn Gln Xaa Ala Gly Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu > PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 52 Glu Glu Glu Xaa Thr Ser Leu Xaa Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Xaa Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Xaa 65 70 75 80 Ile Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro Ser 115 <210> 53 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 53 Glu Glu Glu Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Xaa Ile Leu His Cys Thr 45 Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Cys Xaa Lys Xaa Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Artificial Sequence <220> <223> SiRP alpha variant <400> 54 Glu Glu Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Xaa Arg Xaa Leu 35 40 45 Ile Tyr Asn Gln Xaa Asp Phe Ser Val Arg Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Arg Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 56 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 56 Glu Glu Glu Val Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Gly Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 57 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 57 Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Val Ile Leu His Cys Thr Ile Thr Ser Leu Thr Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Leu Leu 35 40 45 Ile Tyr Asn Gln Arg Glu Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Leu Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 58 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 58 Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Ser Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Gly Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Leu Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 59 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 59 Glu Glu Glu Ile Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Val Ile Ile His Cys Thr Val Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Arg Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Gly Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Val Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 60 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 60 Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ile Ile Leu His Cys Thr Val Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Glu Gly Arg Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Gly Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Leu Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 61 <211> 118 <212 > PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 61 Glu Glu Glu Val Gln Leu Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Glu Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Gly Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 62 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 62 Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Leu Val Ala 1 5 10 15 Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ile Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro Ser 115 <210> 63 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 63 Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Leu Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 64 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SiRP alpha variant <400> 64 Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Lys Gln Gly Pro Phe Pro Arg Val Thr Thr Ile Ser 50 55 60 Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 65 <211> 118 <212> PRT <213> Artificial Sequence <220> < 223> SiRP alpha variant <400> 65 Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Thr Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Gly Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser 115 <210> 66 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 66 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 1 5 10 <210> 67 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 67 Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe 1 5 10 <210> 68 <211> 12 < 212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 68 Cys Arg Arg Arg Arg Arg Arg Glu Ala Glu Ala Cys 1 5 10 <210> 69 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 69 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 Glu Ala Ala Ala Lys Ala Leu Glu Ala Glu Ala Ala Ala Lys Glu Ala 20 25 30 Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala 35 40 45 <210> 70 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 70 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 71 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 71 Gly Gly Gly Gly Gly Gly 1 5 <210> 72 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 72 Gly Gly Gly Gly Ser 1 5 <210> 73 <211> 13 <212> PRT <213> Artificial Sequence < 220> <223> linker peptide <400> 73 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Ala 1 5 10 <210> 74 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 74 Pro Ala Pro Ala Pro 1 5 <210> 75 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 75 Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp 1 5 10 15 Val <210> 76 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 76 Pro Leu Gly Leu Trp Ala 1 5 <210> 77 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 77 Thr Arg His Arg Gln Pro Arg Gly Trp Glu 1 5 10 <210> 78 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 78 Ala Gly Asn Arg Val Arg Arg Ser Val Gly 1 5 10 <210> 79 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 79 Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 80 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 80 Gly Phe Leu Gly 1 < 210> 81 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 81 Gly Ser Ser Gly Gly Ser Gly Ser Ser Gly Gly Ser Gly Gly Gly Asp 1 5 10 15 Glu Ala Asp Gly Ser Arg Gly Ser Gln Lys Ala Gly Val Asp Glu 20 25 30 <210> 82 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> HisX6 tag <400> 82 His His His His His His 1 5 <210> 83 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> FLAG tag <400> 83 Asp Tyr Lys Asp Asp Asp Lys 1 5 <210> 84 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> V5 epitope tag <400> 84 Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 <210> 85 <211 > 8 <212> PRT <213> Artificial Sequence <220> <223> Myc peptide <400> 85 Glu Gln Lys Leu Ile Ser Glu Glu 1 5 <210> 86 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HA tag <400> 86 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 87 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> beta-gal peptide <400> 87 Thr Pro His Pro Ala Arg Ile Gly Leu 1 5 <210> 88 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> AH1 peptide <400> 88 Ser Pro Ser Tyr Val Tyr His Gln Phe 1 5 <210> 89 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> IgG1 Fc domain <400> 89 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 90 <211> 696 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding IgG1 Fc domain <400> 90 gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60 gggggaccgt cagtcttcct cttccccccca a aacccaagg acaccctcat gatctcccgg 120 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300 ggcaaggagt acaagtgcaa ggtct ccaac aaagccctcc cagcccccat cgagaaaacc 360 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480 gacatcgccg tggagtggga gagcaatgg g cagccggaga acaactacaa gaccacgcct 540 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660tacacgcaga agagcctctc cctgtctccg ggtaaa 696

Claims (15)

A fusion protein comprising a Sirpα high-affinity variant consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 15, 17, 19, and 30, and an Fc fragment comprising the hinge region and CH2 and CH3 portions of the heavy chain of the antibody, and A pharmaceutical composition for cancer treatment containing an anthracycline-based anticancer agent as an active ingredient. According to paragraph 1,
A pharmaceutical composition for treating cancer, wherein the Sirpα high-affinity variant consists of the amino acid sequence shown in SEQ ID NO: 5. According to paragraph 1,
The fusion protein is a Sirpα high-affinity variant consisting of a human IgG1 Fc domain consisting of the amino acid sequence shown in SEQ ID NO: 89, a linker peptide consisting of the amino acid sequence shown in SEQ ID NO: 3, and an amino acid sequence shown in SEQ ID NO: 5. A pharmaceutical composition for treating cancer, wherein the anthracycline-based anticancer agent is mitoxantrone. According to paragraph 1,
A pharmaceutical composition for treating cancer, wherein the fusion protein further includes a linker peptide between the Sirpα high-affinity variant and the Fc fragment. According to paragraph 4,
The linker peptides are (G ₄ S) _n , (GSSGGS) _n , KESGSVSSEQLAQFRSLD (SEQ ID NO: 3), EGKSSGSGSESKST (SEQ ID NO: 66), GSAGSAAGSGEF (SEQ ID NO: 67), (EAAAK) _n , CRRRRRREAEAC (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 (SEQ ID NO: 72), AEAAAKEAAAAKA (SEQ ID 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 (SEQ ID NO: 79), GFLG (SEQ ID NO: 80), or GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 81). delete delete According to paragraph 1,
The anthracycline anticancer drugs include daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, mitoxantrone, and sabarubicin. ), or valrubicin, a pharmaceutical composition for treating cancer. delete delete delete delete delete delete delete